Refined Cereal-Based Beverages

ABSTRACT

The present invention relates to methods for preparing cereal-based beverages. Particularly, the present invention provides, for example, methods for steeping and germination of cereal grain under continuous aeration. The present invention also provides for wet milling of germinated cereal grains and direct transfer of the germinated grain, without drying, to the brewery for further processing. Compared to current methods the methods of the present invention significantly reduces water consumption, energy consumption and transport need.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/311,348, filed on Dec. 19, 2018, which is a national phase entrypursuant to 35 U.S.C. § 371 of International Application No.PCT/EP2017/065498, filed Jun. 23, 2017, which claims the benefit ofpriority of European Application No. 16177402.1, filed Jul. 1, 2016,each of which is incorporated by reference herein in its entirety forany purpose.

FIELD OF THE INVENTION

The present invention relates, generally, to germination and preparationof aqueous extracts of cereals (e.g. prepared through mashing),including processes used to produce beer. The invention thus provides alow-input, rapid and continuous process to prepare beer from barleygrain. The methods may be performed in a single facility. The instantinvention is equally applicable to the germination and preparation ofaqueous extracts of other cereal grains—including rice, sorghum, maize,millet and wheat—as well as brewing processes comprising adjuncts.

BACKGROUND OF THE INVENTION

In commercial malting processes, barley grains are germinated, ormalted, under controlled conditions that allow partial mobilization ofthe starch and protein reserves of the starchy endosperm over a periodof 4-6 d. The malting process is typically initiated by immersing thedry barley grain in water. This process is known as steeping where theobjective is not only to clean the grain, but also to raise its moisturecontent to about 40% (w/w) so that the endosperm mobilization step thatfollows will occur more quickly. During steeping, the water is drainedonce to allow re-aeration of the grain. This step is known as the ‘airrest’ and is considered necessary, primarily because the submerged grainbecomes starved of oxygen after about 16 h. After an ‘air rest’ of about8 h, the grain is re-immersed in water to complete the steepingtreatment over another 8-h period—or in a series of re-steeping steps.The two-step steeping process to increase the moisture content of thedry grain to 40%, or higher, takes about 32 h overall. In somemalthouses, spray steeping techniques are used.

The steeped grain is spread for germination, during which enzymessecreted from aleurone and scutellar epithelial cells—together with somethat pre-exist in the starchy endosperm cells—degrade cell walls, starchand protein. Under normal conditions of germination, the phytohormonegibberellic acid (GA) is believed to be synthesized in the nodal region,or elsewhere in the embryo, from where it diffuses along the watergradient (Fincher, 2011).

The maltster usually aims to rapidly induce synthesis of as many of thestarch-degrading enzymes in the grains as possible. In many commercialmalting programs, GA is added to speed up the process of enzymesecretion from the aleurone layer. The starch-degrading enzymes—whichinclude α- and β-amylases, starch debranching enzymes andα-glucosidases—partially depolymerize the starch reserves of the grainto monosaccharides, oligosaccharides, and glucose (Smith et al., 2005;regarding said β-amylases, it is notable that these are deposited in thestarchy endosperm during grain development). The depolymerizationproducts of starch are subsequently used by yeast cells as a carbonsource and are fermented into beer ethanol. Diastatic power is a maltingquality parameter that refers to activity levels of the battery ofstarch degrading enzymes, with high values desirable for brewing.

Other major components of the barley grain include storage proteins,which are also found in the dead starchy endosperm cells and includehordeins as well as water and salt soluble proteins. Depolymerization ofthese also begins naturally in the malting process, but the brewer maymanage the degree of degradation of these proteins so that sufficientpeptides and amino acids are released to support yeast growth during thesubsequent germination step in the brewery. However, if degradation ofstorage proteins proceed too much, the released proteins can causedifficulties in the brewing process. In particular, high levels ofreleased soluble protein can precipitate and form undesirable haze inthe final beer product or increase potential for Strecker aldehydeformation during storage of beer. In specifications of malting quality,an adequate level of free amino nitrogen (FAN) is desirable for yeastgrowth during fermentation. The Kolbach Index is a measure of thesoluble:total protein ratio, with malt giving rise to an adequateKolbach index generally preferred. The extent of protein degradation istherefore an ongoing challenge for the maltster. In addition to the beerprecipitation problem that can be associated with excessive extractedproteins, very high FAN levels can also lead to difficulties, throughthe potential for off-flavour formation.

Maltsters also try to induce high levels of enzymes that degrade cellwall polysaccharides in the barley grain, in particular the(1,3;1,4)-β-glucans and arabinoxylans. Incompletely degraded(1,3;1,4)-β-glucans can be especially troublesome for brewers, becausethese can be extracted from the malt in soluble forms that form highlyviscous aqueous solutions that slow filtration processes in the breweryand contribute to undesirable haze in the final beer. Thus, low levelsof soluble (1,3;1,4)-β-glucan represent an important malting qualityparameter, while high levels of (1,3; 1,4)β-glucanase enzymes remainimportant measures of malt quality.

As noted above, the malting process typically takes about 4-to-5 d.Following the controlled germination steps, the wet malt is dried fromabout a moisture content of 40% to 4-to-5%. This drying process, termedkilning, is very energy consuming and represents a major cost for theindustry.

The kiln-drying has been considered an important part of beer productionfor multiple reasons. One important reason is that during germinationrootlets (also referred to as “culms”) are formed. The rootlets have abitter taste, which affects the aftertaste of beer, and furthermore, therootlets may add undesirable color to beer (see Beer Brewing Technology(1999): 183, published by Shokuhin Sangyo Shimbun as well as U.S. Pat.No. 9,326,542). Once green malt has been kiln drying the rootlets caneasily be removed e.g. using a deculmer. According to the generaltextbook on “Malts and Malting” by D. E. Briggs then “culms must beremoved [ . . . ] since they are extremely hydroscopic, rich in solublenitrogenous substances, contain poorly flavoured and bitter substancesand can be rich in sulphur dioxide and/or nitrosamines. Deculming shouldbe carried out soon after the malt is stripped from the kiln to helpcool it and before the rootlets pick up moisture from the air, becomeslack and pliable (less brittle) and therefore, more difficult to breakand separate” (D. E. Briggs, Malts and Malting; p695 First Edition, 1998Published by Blackie & Professionals, London, ISBNO 412 29800).

Kiln-dried malt generally has a moisture level of 4.5-5.0%. Thekiln-dried malt is subsequently transported from the malthouse to thebrewery by road, rail or sea. This relates to the fact that processes ofmalting and brewing have traditionally been undertaken at differentlocations and often by different corporate entities.

In the brewery, the kiln-dried malt is milled to break open the grain,and the resulting content is extracted with hot water in a process knownas mashing. The extracted material includes partially degraded starch,protein and cell wall molecules as described above, and these arefurther degraded by endogenous grain enzymes that were extracted fromthe malt. At this stage, some brewers add additional—and generallycheaper carbon sources (adjuncts)—to support the subsequent yeastfermentation process and to offset the higher costs of malt. Saidadjuncts can be barley, rice, wheat or other cereal flours fromun-germinated grain, but their addition may necessitate the concomitantaddition of hydrolytic enzymes, because there are insufficientendogenous enzymes in the malt to degrade the components of the adjunct.The added enzymes are usually from unpurified and relatively cheapextracts of fungal and/or bacterial cultures. The addition of exogenousenzymes is not legal in some countries, particularly where beer must beproduced under tightly regulated settings.

Further degradation of the starch, and other endosperm componentsextracted in hot water, proceed in a process known as saccharification.Following mashing, the extracts are filtered, often in a lauter tun, andcooled. The extract may be boiled in the presence of hops or hopextracts, and upon cooling yeast cultures are added for the fermentationof released sugars to ethanol. The beer so produced is usually maturedand filtered before bottling. The beer may also be carbonated prior tobottling.

SUMMARY OF THE INVENTION

The instant invention provides methods for fast germination andpreparation of aqueous extracts of cereals. The methods significantlyspeed up the process of preparing wort for production of cereal-basedbeverages, while maintaining the potential for preparing said wort withhigh levels of fermentable sugars, preferably with low levels ofβ-glucan and xylan. In particular, the beverages prepared from said wortmay be characterized by a low level of astringent taste.

The invention demonstrates that a continuous and integrated brewingprocess going from dry barley grain to beer can be undertaken at asingle location. In that respect, the present invention provides methodsfor combined steeping and germination of cereal grain to a moisturecontent of for example >30%, preferably >35% through constant aeration.The invention may thereby provide for significant water savings throughthe elimination of drying of the malt as well as significant energysavings e.g. by omission of the kiln drying step.

In the present invention, the grains are submerged and incubated in anaqueous solution (typically water), wherein O₂ is supplied to saidliquid. Typically, said O₂ is supplied continuously throughout theincubation, which for example may be for in the range of 20 to 72 h andin general allow the grain not only to reach the appropriate moisturecontent but also to germinate in a controlled manner. The step ofgermination may also comprise one or more air-rests typically followingthe step incubation in aqueous solution under aeration. This controlledgermination process may be shortened by the addition of one or morecompound capable of accelerating the germination. For example, thephytohormone gibberellic acid (GA) may be added to the aqueousliquid—either from the onset or during incubation. GA ‘activates’ geneexpression in its target cells, namely the aleurone and the scutellarepithelium of the barley grain, including genes encoding endogenousenzymes necessary for the hydrolysis of starch, storage proteins andcell wall polysaccharides. Thus, the total time required for steepingand germination may be reduced from more than 5 d in conventionalbrewing processes to ˜2 d or less using the present invention.

Following the combined steeping and germination processes, the methodsof the present invention comprise a step of finely dividing thegerminated cereal grains. A particularly interesting aspect of theinvention is that the methods of the invention allow proceeding withfinely dividing the germinated cereal grains immediately after thegermination. Accordingly, the methods in general do not comprise a stepof drying the germinated cereal grains. In particular, the methods donot comprise a step of kiln drying the germinated cereal grains. Asdescribed above, one important aspect of kiln drying is to allow easyremoval of rootlets. Prior to drying removal of rootlets is difficult toachieve. However, the germinated cereal grains prepared according to themethods of present invention have significantly reduced rootlets(typically less than 4 g per 100 g germinated barley (dry weight)), andas shown by the present invention, the step of kiln drying is notrequired in respect of cereal germinated according to the methods of theinvention.

The germinated cereal grains may for example be finely divided bysubjecting the germinated cereal grains to wet milling, followed by astep of preparing an aqueous extract, for example, by mashing at apredetermined temperature for any suitable time as described hereinbelow in the section “Preparing an aqueous extract”. The conversion ofreleased saccharides, e.g. polysaccharides, and proteins can befacilitated during mashing by the addition of mixtures of exogenousenzymes that catalyze the degradation of starch, storage proteins andcell wall polysaccharides. The enzymes can be partially purified frombarley itself, from malt or from other sources—or, alternatively, fromfungal and/or bacterial enzyme mixtures that can be purchased fromcommercial sources.

Aside from benefits as described above, the present invention obviatesnot only the need for kiln-drying the malt, but also the need fortransporting the dried grain from the malthouse to the brewhouse. Energycost savings of up to 50% can be achieved through this combined steepingand germination invention, which may greatly reduce the carbon footprintof the industry. This is of importance because of world-wide, increasinglegislative and taxation pressures in most countries to reduce carbonfootprints of the malting and brewing industries.

Further in the context of sustainability, the present invention allowsthe entire production of beer to be performed in already-existingbrewery equipment, so that little additional capital expenditure isrequired.

The invention is further defined in the claims attached hereto.

DESCRIPTION OF DRAWINGS

FIG. 1 shows an example of equipment useful for performing the method ofthe invention in which grains can be immersed in an aqueous solution andaerated continuously. The equipment comprises an inlet for cereal grains(1), a tank, e.g. a steeping tank (2); inlets for gas, e.g. sinterstones (3); a pump e.g. an air pump (4); an outlet for cereal grains(5); a grain pump (6); an equipment for finely dividing cereal grainse.g. a mill (7); an inlet (8); a vessel, e.g. a mashing vessel (9), and;an outlet (10).

FIGS. 2A-2B show hull-less barley grains after incubation in water at15° C. or 25° C. for either 24 h or 48 h under the indicated airflow.FIG. 2A shows a collection of grains, whereas FIG. 2B shows individualrepresentative grains. It is notable that already after 24 h at 15° C.,the grains had initiated germination (with a visible chit even with only30 L/h airflow, and several small rootlets were visible after 48 h.After 24 h at 25° C., the grains had initiated germination and containeda visible chit, even with an airflow of only 30 L/h. With a higherairflow, or after 48 h, several small rootlets were visible.

FIGS. 3A-3B show hulled barley grains after incubation in water at 15°C. or 25° C. for either 24 h or 48 h under the indicated airflow. FIG.3A shows a collection of grains, whereas FIG. 3B shows individualrepresentative grains.

FIGS. 4A-4C show diagrams related to activities of α-amylase (FIG. 4A),β-amylase (FIG. 4B) and limit dextrinase (panel 4C) in grains germinatedin the presence of various amounts of GA in water under aeration.

FIG. 5 shows an example of a mashing process with steeped, germinatedand wet-milled grains.

FIG. 6 shows a diagram of the filterability of wort, which had beenprepared from grains germinated under varying aeration in the presenceor absence of the brewing enzyme mixture Ultraflo Max (UFM).

FIG. 7 shows a diagram on the level of fermentable sugars in wortprepared from grains germinated for 48 h at 25° C. under varyingaeration conditions. Samples supplemented with the brewing enzymemixture Ultraflo Max are marked with UFM.

FIG. 8 shows an example of equipment useful for performing the steps ofthe invention in which grains can be immersed in an aqueous solution andaerated continuously. The equipment comprises: an inlet for cerealgrains (1); a tank, e.g. a FlexTank (2); a grid or a mesh, e.g. a metamesh (3); inlets for gas, e.g. sinter stones (4) and; a pump, e.g. anair pump (5).

FIG. 9 shows α-amylase, β-amylase and limit dextrinase activity in 4commercially available wheat varieties 1—Benchmark, 2—Creator,3—Pistoria, 4—Sheriff in wheat (white columns), after 24 h (greycolumns) or 48 h (black columns).

FIG. 10 shows α-amylase in 8 barley varieties after germination underaeration (1—Alexis, 2—Chief, 3—Chill, 4—Paustian, 5—Planet, 6—Prestige,7—Quench, 8—Tipple)

FIG. 11 shows α-amylase, β-amylase and limit dextrinase activity inhulled barley peeled to remove husk and subsequently germinated underaeration.

FIGS. 12A and 12B show α-amylase, β-amylase and limit dextrinaseactivity in barley germinated in aqueous solution under aeration (WA)with or without air-rest(s) (A) for the times indicated in the barleyhull-less 01 (FIG. 12A) and in the barley hulled 02 (FIG. 12B).

FIG. 13 shows the % weight loss after rootlet removal in both hulled andhull-less barley germinated by incubation in aqueous solution underaeration for 48 h (48 hWA) and barley germinated by standard steepingconditions for 96 h (Malting 96 h).

FIG. 14 shows the NDMA content in ungerminated barley, in barleygerminated by the methods of the invention (Malt 1a), and in 3 differentconventional malts (Malt 1b, Malt 2 and Malt 3).

DEFINITIONS

The term “approximately” when used herein in relation to numericalvalues preferably means ±10%, more preferably ±5%, yet more preferably±1%.

The term “adjunct” as used herein refers to carbon-rich raw materialsources added during preparation of beer. The adjunct may be anungerminated cereal grain, which may be milled together with thegerminated grains prepared according to the invention. The adjunct mayalso be a syrup, sugar or the like.

The term “chit” as used herein refers to the embryonic growing bud thatis visible during the germination phase of a cereal grain.

The term “water content” of a grain as used herein refers to the % ofwater w/w in said grain.

The term “germinated grain” as used herein refers to grains havingdeveloped a visible chit, preferably a chit of at least 1 mm, such as ofat least 2 mm.

The term “3-glucan” as used herein, unless otherwise specified, refersto the cereal cell wall polymer “(1,3;1,4)-β-glucan”.

Similarly, the term “xylan” as used herein, unless otherwise specified,refers to the cereal cell wall polymer “arabinoxylan”.

The terms “kiln dried malt” and “kilned malt” as used herein refer togerminated cereal grains, which have been dried by kiln drying.

The term “sprouted grain” as used herein refers to a grain havingdeveloped a visible chit and a visible stem.

The term “steeping” as used herein refers to the process of increasingthe water content of a cereal kernel.

The term “3-glucanase” as used herein refers to enzymes with thepotential to depolymerize cereal β-glucan. Accordingly, unless otherwisespecified, the term “3-glucanase” refers to an endo- or exo-enzyme ormixture thereof characterized by (1,3;1,4)-β- and/or (1,4)-β-glucanaseactivity.

The term “xylanase” as used herein refers to enzymes with the potentialto degrade main and side chains of xylan and arabinoxylan. Accordingly,unless otherwise specified, the term “xylanase” refers to an enzyme oran enzyme mixture characterized by enzymic activities derived by one ormore of the following enzyme classes: endo-1,4-xylanase;exo-1,4-xylanase; arabinofuranosidase; ferulic acid esterase.

Enzyme activities of cereal grains as used herein refer to theactivities measured in flour prepared from the specified grain type. Forexample, 10 U/g of α-amylase activity per gram cereal grain refers tosaid α-amylase activity (10 U) measured in an aqueous extract derivedfrom 1 g of flour (dry matter) from said cereal. α-amylase activity isdetermined by K-CERA 01/12 (protocol and kit available from Megazyme,Ireland). β-amylase activity is determined by the K-BETA3 (protocol andkit available from Megazyme, Ireland). Limit-dextrinase activity isdetermined by the T-LDZ1000 (protocol and kit available from Megazyme,Ireland).

The volume of a gas as indicated herein refers to the volume of said gasat 1 atm and 20° C.

The volume of O₂ as indicated herein refers to the volume of 02 at 1 atmand 20° C. In embodiments of the invention where 02 is comprised in amixture of gasses, then the total volume of the gas mixture may bedetermined, and the volume of 02 may be calculated as the percentage ofthe total volume constituted by O₂. By way of example then atmosphericair comprises 21% O₂. Thus the volume of 02 within atmospheric air asused herein is 21% of the total volume of atmospheric air.

DETAILED DESCRIPTION Cereal Grains

The methods of the present invention comprise a step of germination,which comprises incubating cereal grains in an aqueous solution. Itshould be noted that the mixture of aqueous solution and cereal grainsmay be considered a suspension.

The cereal grain may be the grain of any cereal, for example a cerealselected from the group consisting of barley, rice, sorghum, maize,millet, triticale, rye, oat and wheat. In preferred embodiments of theinvention the cereal grains are barley grains. Said grains may be grainsof any barley variety, such as any of the barley varieties describedherein below in the section “Barley”.

The cereal grains may have a relatively low water content beforeincubation in said aqueous solution. For example, the cereal grains mayhave a water content of at the most 30%, preferably at the most 20%,such as at the most 15%, for example in the range of 5 to 15%.

Before incubation in said aqueous solution, the cereal grain may havebeen subjected to one or more steps of antimicrobial treatment. Saidantimicrobial treatment may be any useful antimicrobial treatment, whichdoes not impair the grains potential for germination. The antimicrobialtreatment could for example be treatment with one or more antimicrobialagents. Said antimicrobial agents may be any antimicrobial agent, whichat the concentrations used is not toxic to cereal grains. For example,the antimicrobial agent may be a chlorine containing compound, e.g.hypochlorite. The antimicrobial agent may also be peroxide, e.g.hydrogen peroxide and/or peracetic acid. Non-limiting examples of usefulcommercial antimicrobial agents include P3-Hypochloran®, P3-Peroxysan®or P3-oxonia active 150®. Cereal grains may be treated with hypochloranat a concentration in the range of 0.1 to 10%, such as in the range of0.5 to 5%, for example approximately 1%, such as 1%. The cereal grainsmay be treated with said hypochloran for in the range of 15 min to 10 h,such as in the range of 1 to 5 h, for example for in the range of 2 to 4h. After treatment, the cereal grains may be washed one or more times.

In some embodiments of the invention, the antimicrobial treatment isperformed by incubating cereal grains in an aqueous solution comprisingthe antimicrobial agent. Immediately after said incubation, the step ofgermination may be initiated, e.g. by initiating aeration. Thus, in suchembodiments, it is not required to change the aqueous solution, and thesame aqueous solution may be used for the antimicrobial treatment and atleast during the onset of the subsequent step of germination. This mayin particular be the case, when the antimicrobial agent is a peroxide,e.g. hydrogen peroxide.

It may be preferred that said cereal grains have not been subjected togermination prior to the incubation in aqueous solution according to theinvention. Accordingly, it may be preferred that the cereal grains havenot been subjected to a step of pre-germination.

As described above the cereal grain may be any cereal grain. Some cerealgrains comprises a hull, whereas other cereal grains are hull-less.Hulled cereal grains may be treated to remove at least part of the hullprior to the step of germination. In general, treatment of remove hullis not required if a hull-less cereal grain is used. Hull-less cerealsinclude for example hull-less barley varieties and wheat.

Hulled cereal grains may be treated to remove hull by subjecting thecereal grains to physical treatment removing hull. Said physicaltreatment may for example be selected from the group consisting ofpolishing, sanding, peeling and smoothening. Preferably, the physicaltreatment results in a loss of the hull. Loss of the hull may bedetermined as an overall weight loss. Thus, the physical treatmentpreferably leads to a loss of in the range of 1 to 4%, such as in a lossof in the range of 1.5 to 3.0% of the total weight of the cereal grains.

Germination

The methods of the present invention comprise a step of germination ofcereal grains. The step of germination comprises a step of incubatingcereal grains in an aqueous solution typically under aeration.

Incubation in an Aqueous Solution Under Aeration

The cereal grains may be any of the cereal grains described herein abovein the section “Cereal grains”, and the aqueous solution may be any ofthe aqueous solutions described herein below in the section “Aqueoussolution”.

It may be preferred that during said incubation in aqueous solution,then the cereal grains are completely covered by said aqueous solutionduring the entire incubation. Thus, the cereal grains may for example beincubated in at least 1, preferably at least 1.5, more preferably atleast 2, for example in the range of 1 to 10, such as in the range of 1to 5, for example in the range of 1.5 to 3 L aqueous solution per kgcereal grain (dry weight).

Thus, in some embodiments, the cereal grains are submerged in theaqueous solution during the entire incubation.

In other embodiments, the cereal grains take up the aqueous solution ina manner, so that at the end of the incubation on said aqueous solutionall of said aqueous solution is taken up by the cereal grains.

In other embodiments, aqueous solution remaining after incubation underaeration may be drained off the cereal grains.

In some embodiments, it is preferred that after incubation of the cerealgrains in said aqueous solution, then the majority, for example at least70%, preferably at least 80%, more preferably at least 90%, even morepreferably, at least 95%, such as essentially all of the cereal grainscontain a visible chit of at least 1 mm.

In some embodiments, after incubation of the cereal grains in saidaqueous solution, then the majority, for example at least 70%, such asat least 80%, for example at least 90%, such as at least 95%, such asessentially all of the cereal grains contain one or more visiblerootlets.

In some embodiments, after incubation of the cereal grains in saidaqueous solution, then the majority, for example at least 70%, such asat least 80%, for example at least 90%, such as at least 95%, such asessentially all of the cereal grains contain one or more visiblerootlets and a visible stem.

In some embodiments, after incubation of the cereal grains in saidaqueous solution, then the cereal grains have a water content of atleast 30%, preferably at least 35%, more preferably of at least 37%, forexample in the range of 35 to 60%, such as in the range of 35 to 50%,for example in the range of 37 to 60%, such as in the range of 37 to50%.

The water content of cereal grains may be determined by determining theweight of the cereal grains, followed by drying said cereal grains anddetermining the weight of the dried cereal grains. The difference inweight of the wet and dry cereal grains is considered to be water, andthe water content is provided as the weight of the water divided by thetotal weight of the cereal grains (wet cereal grains). The water contentprovided in % is thus a w/w %.

The cereal grains may be incubated in said aqueous solution forsufficient time to allow germination of the majority of said cerealgrains as described above. The cereal grains may also be incubated insaid aqueous solution for sufficient time in order to obtainaforementioned water content. In some embodiments of the invention, thestep of germination comprises an incubation in the aqueous solutionfollowed by an air-rest. In such embodiments the incubation in aqueoussolution is performed for a time sufficient to allow sufficient enzymeactivity in the germinated cereal grains after the air-rest. The enzymeactivity is preferably as described herein below in the section“Germinated cereal grains”.

Typically, the cereal grains are incubated in the aqueous solution forat least 20 h, such as at least 24 h. Typically, the grains areincubated in said aqueous solution for at the most 72 h, such as for atthe most 60 h, for example for at the most 48 h. Thus, in someembodiments the cereal grains are incubated in said aqueous solution forin the range of 20 to 72 h, such as for in the range of 20 to 60 h, forexample for in the range of 20 to 48 h, for example for in the range of20 to 30 h, such as for in the range of 22 to 26 h.

In some embodiments the cereal grains are incubated in the aqueoussolution under aeration for in the range of 24 to 60 h, preferably forin the range of 40 to 55 h, more preferably for in the range of 45 to 50h. This may in particular be the case in embodiments of the invention,where germination does not comprise a step of air-rest, for example inembodiments, wherein the cereal grains are being finely dividedessentially immediately following the incubation in the aqueoussolution.

In some embodiments the cereal grains are incubated in the aqueoussolution under aeration for in the range of 24 to 70 h, preferably forin the range of 40 to 60 h, more preferably for in the range of 45 to 55h. This may in particular be the case in embodiments of the invention,where germination does not comprise a step of air-rest, for example inembodiments, wherein the cereal grains are being finely dividedessentially immediately following the incubation in the aqueoussolution.

In some embodiments the cereal grains are incubated in the aqueoussolution under aeration for in the range of 16 to 40 h, preferably forin the range of 16 to 30 h, such as in the range of 20 to 30 h, morepreferably for in the range of 22 to 26 h. This may in particular be thecase in embodiments of the invention, where germination furthercomprises a step of air-rest.

The cereal grain may be incubated at any useful temperature, however itmay be preferred that incubation is performed at a temperaturesufficiently high to allow fast increase in water content. As shownherein below in Example 1, then an increase in the temperaturesignificantly may advance increase in water content. Thus, it may bepreferred that the cereal grains are incubated in said aqueous solutionat a temperature of at least 15° C., such as at least 20° C., such as atleast 25° C. In particular, the cereal grains may be incubated at in therange of 10 to 35° C., preferably in the range of 15 to 30° C., such asin the range of 20 to 30° C., for example in the range of 25 to 30° C.

In particular, in embodiments of the invention wherein the cereal grainsare incubated at a temperature in the range of 20 to 30° C., then saidcereal grains may be incubated for in the range of 20 to 48 h.

As described herein above, then said cereal grains are frequentlyincubated in said aqueous solution, while O₂ is passed through theaqueous solution. This is also referred to as said cereal grains beingincubated in the aqueous solution under aeration. Preferably, O₂ isbeing passed through the aqueous solution continuously during the entireincubation. Said O₂ may be passed through the aqueous solution in anyuseful manner, however frequently a gas containing O₂ is introduced atthe bottom and/or in the lower part of the container comprising theaqueous solution with the cereal grains. Typically, the gas will diffusethrough the aqueous solution/cereal grain mixture and leave the aqueoussolution/cereal grain mixture from the top of the aqueous solution. Inparticular, the incubation may be performed in an apparatus as describedherein below in the section “Apparatus”. It is also possible that heavygasses, notably CO₂, are withdrawn from the bottom of the container,whereby fresh air/O₂ may be provided from the upper part of thecontainer.

Said O₂ may be added to said aqueous solution as a pure O₂. Frequently,however, said O₂ is comprised within a gas mixture. In one embodimentsaid O₂ is comprised within atmospheric air. Thus, the method of theinvention may comprise passing atmospheric air through said aqueoussolution.

In general, at least 2 L, preferably at least 3 L, more preferably atleast 4 L, yet more preferably at least 5 L, even more preferably atleast 6 L O₂ passes through said aqueous solution per kg cereal grainsper h. The weight of said cereal grains is the dry weight. For example,in the range of 2 to 100 L, for example in the range of 2 to 75 L, suchas in the range of 2 to 50 L, for example in the range of 4 to 100 L,for example in the range of 4 to 75 L, such as in the range of 4 to 50L, for example in the range of 6 to 100 L, for example in the range of 6to 75 L, such as in the range of 6 to 50 L O₂ passes through saidaqueous solution/cereal grain mixture per kg cereal grains (dry weight)per h.

In one embodiment it is preferred that at least 20 g O₂ per kg cerealgrain, more preferably at least 30 g O₂ per kg cereal grain, yet morepreferably at least 40 g O₂ per kg cereal grain, for example in therange of 40 to 100 g O₂ per kg cereal grain, such as in the range of 40to 80 g O₂ per kg cereal grain, for example in the range of 60 g O₂ perkg cereal grain is passed through said aqueous solution/cereal grainmixture per h. The weight of the cereal grain is provided as dry weight.During incubation the cereal grains typically take up at least some ofthe aqueous solution and accordingly the concentration of O₂ in theaqueous solution will typically vary during the incubation. Typically,the amount of O₂ supplied per L aqueous solution per h is in the rangeof 40 to 200 g, preferably in the range of 50 to 150 g.

As noted above, it is frequently atmospheric air that is passed throughthe aqueous solution. Thus, the method may comprise passing at least 10L, preferably at least 15 L, more preferably at least 20 L, yet morepreferably at least 25 L, even more preferably at least 30 L atmosphericair through said aqueous solution per kg cereal grains per h. The weightof said cereal grains is the dry weight. For example, in the range of 10to 500 L, for example in the range of 10 to 375 L, such as in the rangeof 10 to 250 L, for example in the range of 20 to 500 L, for example inthe range of 20 to 375 L, such as in the range of 20 to 250 L, forexample in the range of 30 to 500 L, for example in the range of 30 to375 L, such as in the range of 30 to 250 L atmospheric air is passedthrough said aqueous solution per kg cereal grains (dry weight) per h.In one embodiment in the range of 50 to 110 L, preferably 80 to 100 Latmospheric air is passed through said aqueous solution per kg cerealgrains (dry weight) per h.

Air-Rest

In addition to said incubation in aqueous solution under aeration, thecereal grains may also be incubated in air (e.g. in the absence ofaqueous solution). The step of incubation in air may also be referred toas “air-rest”. Thus, after incubation in aqueous solution underaeration, remaining aqueous solution may be drained off and the cerealgrains may be incubated in air. Alternatively, after incubation inaqueous solution under aeration, all aqueous solution has been taken upby the cereal grains, which may then be incubated in air. Saidincubation in air is preferably performed under aeration, for example O₂may be passed through the container comprising the cereal grains.Preferably, O₂ is being passed through said container during the entireair-rest. The amount of O₂ being passed through the container comprisingthe cereal grains, may be the same amount of O₂ as being passed throughthe aqueous solution as described above. The O₂ may be provided in theform of atmospheric air.

The air-rest may be performed for any suitable amount of time,preferably for in the range of 18 to 50 h, more preferably for in therange of 18 to 38 h, for example for in the range of 22 to 35 h.

During the air-rest additional water or aqueous solution may be added tothe cereal grains, e.g. by irrigation or sprinkling. However, during theair-rest, the cereal grains should not be submerged in aqueous solution.

Germination Methods

The germination may also comprise several steps of incubation in aqueoussolution and/or several steps of air-rest. In general the first step isa step of incubation of cereal grains in an aqueous solution underaeration as described above. Thus, the germination may comprise orconsist of the following steps:

-   -   incubation of cereal grains in an aqueous solution under        aeration as described above in the section “Incubation in an        aqueous solution under aeration”    -   incubation of cereal grains in air as described above in the        section “Air-rest”

In this embodiment, the incubation of cereal grains in an aqueoussolution under aeration may for example be performed for in the range of16 to 30 h, such as in the range of 20 to 30 h, for example in the rangeof 22 to 26 h, whereas the air-rest may be performed for in the range of18 to 38 h, for example in the range of 22 to 35 h.

The germination may also comprise or consist of the following steps:

-   -   incubation of cereal grains in an aqueous solution under        aeration as described above in the section “Incubation in an        aqueous solution under aeration”    -   incubation of cereal grains in air as described above in the        section “Air-rest”    -   incubation of cereal grains in an aqueous solution under        aeration as described above in the section “Incubation in an        aqueous solution under aeration”

The germination may also comprise or consist of the following steps:

-   -   incubation of cereal grains in an aqueous solution under        aeration as described above in the section “Incubation in an        aqueous solution under aeration”    -   incubation of cereal grains in air as described above in the        section “Air-rest”    -   incubation of cereal grains in an aqueous solution under        aeration as described above in the section “Incubation in an        aqueous solution under aeration”    -   incubation of cereal grains in air as described above in the        section “Air-rest”

The germination may also comprise or consist of the following steps:

-   -   incubation of cereal grains in an aqueous solution under        aeration as described above in the section “Incubation in an        aqueous solution under aeration”    -   incubation of cereal grains in air as described above in the        section “Air-rest”    -   incubation of cereal grains in an aqueous solution under        aeration as described above in the section “Incubation in an        aqueous solution under aeration”    -   incubation of cereal grains in air as described above in the        section “Air-rest” incubation of cereal grains in an aqueous        solution under aeration as described above in the section        “Incubation in an aqueous solution under aeration”

The time for each incubation may vary, however typically the entire stepof germination, i.e. the total time for all incubations in aqueoussolution and all air-rests does not exceed 72 h, more preferably doesnot exceed 60 h, even more preferably does not exceed 54 h. Thus, it maybe preferred that the entire step of germination is performed for in therange of 20 to 72 h, such as for in the range of 24 to 60 h, for examplefor in the range of 24 to 48 h. Accordingly, if the germinationcomprises several steps of incubation in aqueous solution and/orair-rests, then each incubation step is generally shorter.

It is preferred that all steps of the germination is performed withinthe same container. Said container may in particular be a tank, such asany of the tanks described herein below in the section “Apparatus”.

In some embodiments one or more exogenous enzymes may be added. Forexample, one or more enzymes may be added during the step of germinatione.g. as described in WO2016/071463.

The cereal grains are preferably finely divided essentially immediatelyafter germination. Accordingly, the methods of the invention dopreferably not comprise a step of drying between the step of germinationand finely dividing the cereal grains.

Germinated Cereal Grains

The invention relates to a method comprising a step of producinggerminated cereal grains.

The germinated cereal grains preferably comprise one or more hydrolyticenzyme activities, for example provided by α-amylases, β-amylases,starch debranching enzymes (such as limit dextrinases), α-glucosidasesand proteases.

Frequently, the onset of hydrolytic enzyme activity may be occurring ina timely coordinated manner, and thus the activity of some hydrolyticenzymes may be used as a marker for other hydrolytic enzyme activities.

Accordingly, it is preferred that the germinated cereal grains have anadequate level of measurable α-amylase activity. Preferably, thegerminated cereal grains have a measurable α-amylase activity of atleast 4 U/g, such as at least 10 U/g cereal grain (dry weight). Thus,preferably the germinated cereal grains may have a measurable α-amylaseactivity of at least 20 U/g, for example of at least 30 U/g, such as atleast 40 U/g, for example at least 50 U/g cereal grains (dry weight). Inembodiments of the invention where the cereal grain is wheat theα-amylase activity may be at least 30 U/g. In some embodiments of theinvention, the cereal grain may have an α-amylase activity of at least50 U/g, for example of at least 60 U/g, such as at least 70 U/g cerealgrains (dry weight). This may in particular be the case in embodimentswhere the cereal grains are incubated in an aqueous solution underaeration followed by an air-rest.

The α-amylase activity is preferably determined according to standardmethods, e.g. by using the Ceralpha kit (K-CERA) from Megazyme, Ireland.In particular, the α-amylase activity may be determined as described inExample 2 below.

It may also be preferred that the germinated cereal grains have anadequate level of measurable β-amylase activity. Preferably, thegerminated cereal grains have a measurable β-amylase activity of atleast 5 U/g cereal grain (dry weight). Thus, preferably the germinatedcereal grains may have a measurable β-amylase activity of at least 10U/g, for example at least 15 U/g cereal grains (dry weight).

Preferably, the β-amylase activity is determined according to standardmethods, e.g. by using the Betamyl kit (K-BETA3) from Megazyme, Ireland.In particular, the β-amylase activity may be determined as described inExample 2 below.

It is also preferred that the germinated cereal grains have an adequatelevel of limit dextrinase activity. Preferably, the germinated cerealgrains have a limit dextrinase activity of at least 5 mU/g cereal grain(dry weight). Thus, preferably the germinated cereal grains may have alimit dextrinase activity of at least 10 mU/g, for example of at least15 mU/g, such as at least 20 mU/g cereal grains (dry weight). Inembodiments of the invention where the cereal grain is wheat the limitdextrinase activity may be at least 8 U/g. In some embodiments of theinvention, the cereal grain may have a limit dextrinase activity of atleast 20 U/g, for example of at least 22 U/g, such as at least 25 U/gcereal grains (dry weight). This may in particular be the case inembodiments where the cereal grains are incubated in an aqueous solutionunder aeration followed by an air-rest.

Preferably the limit dextrinase activity is determined according tostandard methods, e.g. by using the Limit Dextrizyme kit T-LDZ1000 fromMegazyme, Ireland. In particular, the limit dextrinase activity may bedetermined as described in Example 2 below.

Interestingly, the germinated cereal grains according to the inventionhave significantly reduced rootlets compared to conventional green malt.Thus, the germinated cereal grains according to the invention preferablycontains at the most 4 g rootlets per 100 g germinated barley,preferably at the most 3 g rootlets per 100 g germinated barley, evenmore preferably at the most 2 g rootlets per 100 g germinated barley,for example at the most 1.1 rootlets per 100 g germinated barley,wherein both the mass of the rootlets and the mass of the germinatedbarley is provided as dry weight. The mass of rootlets is preferablydetermined as described in Example 10 below.

Nitrosamines (NDMA) are chemical compounds of the chemical structureR¹N(—R²)—N═O, that is, a nitroso group bonded to an amine. Mostnitrosamines are carcinogenic. Even though the levels of nitrosamines inmodern malts are low, the germinated barley kernels according to theinvention never-the-less still have a significantly reduced content ofNDMA compared to conventional malt. In one embodiment, the germinatedbarley kernels according to the invention comprise at the most 0.15μg/kg N DMA, preferably at the most 0.12 μg/kg NDMA, for example at themost 0.10 μg/kg NDMA.

Aqueous Solution

The aqueous solution may be any aqueous solution. The aqueous solutionmay be considered a solution even though the mixture of aqueous solutionand cereal grains may be regarded as a suspension. Frequently, theaqueous solution is water, such as tap water. One or more additionalagents may be added to said water, and thus the aqueous solution may bewater, such as tap water comprising one or more additional agents. Saidadditional agents may be comprised in the aqueous solution from theonset or they may be added during the incubation.

Said additional agents may, for example, be compounds capable ofaccelerating germination of cereal grains. Thus, the aqueous solutionmay comprise gibberellic acid (GA), for example the aqueous solution maycomprise GA at a concentration of at least 100 nM, for example at aconcentration of at least 1000 nM, such as at a concentration of in therange of 100 to 100,000 nM, for example at a concentration of in therange of 500 to 2000 nM. Said GA may be present in the aqueous solutionfrom the onset of incubation, or it may be added during incubation. SaidGA may any GA, e.g. GA₃ or GA₇. In one embodiment said GA is GA₃.

The additional agent may also be an antifoaming agent. Said antifoamingagent may for example be any food grade antifoaming agent, for exampleFoamzol FCD511 (AB Vickers, UK).

Apparatus

The methods of the invention may be performed using one or moreapparatuses suitable for performing the methods.

For example, the step of incubating cereal grains in an aqueous solutionmay be performed in a container equipped with one or more air pumps. Thecontainer may be any container in which cereal grains can be incubatedin an aqueous solution. In some embodiments, the container may be atank, e.g. a tank as described below.

One example of an apparatus useful for incubating cereal grains isprovided in FIG. 8 . The apparatus comprises a tank (2), which shouldhave a sufficient volume to comprise the cereal grains and the aqueoussolution. The tank shown in FIG. 8 is cylindrical, but the tank to beused with the invention may have any suitable shape, for example it maybe a cylindrical tank, for example a cylindrical tank having a conicalbottom part. The tank may be prepared from any suitable material, e.g.plastic (such as Plexiglas) or metal (e.g. stainless steel or copper).

The tank comprises at least one inlet (1) for cereal grains, which canbe used to add cereal grains to the tank. The inlet may also be used foradding other compounds to the tank, e.g. the inlet may be used foradding the aqueous solution, e.g. water. The inlet may be positioned atany useful position in the tank, and in some embodiments the inlet ispositioned in the upper part of the tank, e.g. at the top of the tank.The inlet should be of a sufficient size to allow addition of cerealgrains. Even though not required, the tank may comprise additionalinlets in addition to the inlet for cereal grains.

The tank may optionally comprise a grid or mesh (3) positionedessentially horizontally in the tank. If present, such as grid or meshis typically positioned in the lower ⅓, such as in the lower ⅕, of thetank. The grid or mesh preferably only contains openings, which aresmaller than the cereal grains. The grid or mesh may be made from anysuitable material, such as plastic or metal, and may for example be ametal mesh. Thus, the grid or mesh may be used to separate the cerealgrains from the bottom part of the tank. However, the tank willfrequently not comprise a mesh. In particular, if the tank comprise anoutlet for cereal grains in the bottom or close to the bottom, then saidtank in general do not comprise a grid of mesh.

Furthermore, the tank comprises one or more inlets for gas (4). Saidinlets may be any inlet through which a gas comprising O₂ can be passedinto the tank. The inlets for gas may have a shape, which allows the gasto enter the aqueous solution at a high speed ensuring diffusion of thegas through the aqueous solution. Thus, for example the inlets for gasmay be nozzles, jets, diffusion stones or sinter stones. In oneembodiment the inlets for gas are sinter stones. The inlets for gas arein general connected to a pump (5), which pump gas into the tank,through said inlets. The pump may be any pump capable of pumping gas,e.g. air through the inlets for gas. It is preferred that the tankcomprises multiple inlets for gas, e.g. at least 2, such as at least 3,for example in the range of 3 to 20. The inlets for gas may bepositioned at any position in the tank, but usually, they are positionedin the bottom ⅓, such as in the bottom ⅕ of the tank. This allows gas toenter the aqueous solution from the bottom and to diffuse upward throughthe aqueous solution. Excess gas may leave the tank through any openingin the tank, e.g. through the inlet for adding cereals. In oneembodiment it may be preferred that said inlets (3) are positioneddirectly on the side walls of the tank (2), preferably on the lower partof the side walls, e.g. as shown in FIG. 1 .

An example of an apparatus useful for performing several steps of themethods of the invention is shown in FIG. 1 . The apparatus comprises aninlet for cereal grains (1), a tank (2), inlet(s) for gas (3) and a pump(4), which may be any of the inlets for cereal grains, tanks, inlets forgas and pumps described herein above in relation to FIG. 8 . The tank(2) may comprise an outlet for cereal grains (5) positioned in the lower⅓, such as lower ⅕ of the tank, e.g. positioned at the bottom of thetank. Said outlet may be used both for removing cereal grains as well asfor removal of other components kept in the tank, e.g. the aqueoussolution. Said outlet may be connected to a grain pump (6), e.g. viatubing. Said grain pump (6) may be any pump capable of pumping grainsfrom the tank (2) to the equipment for finely dividing the cereal grains(7) and optionally further to the mashing vessel (9).

The apparatus comprises an equipment for finely dividing the cerealgrains (7). Said equipment may be any equipment capable of finelydividing cereal grains having a water content of above 20%, for exampleabove 35%. The equipment may in particular be a grinder or a mill, forexample a wet mill. The equipment (7) may be connected to the tank (2)and to the vessel (9) by tubing(s). Movement of grains from the tank (2)to the equipment (7) and further to the vessel (9) may be ensured by apump (6).

The apparatus may also comprise a vessel (9). The vessel (9) may be anyvessel, which can comprise a aqueous extract and which can withstandtemperatures used for mashing, e.g. temperatures of up to 90° C., suchas up to 85° C., for example up to 80° C. Thus, the vessel can be madeof any material tolerating such temperatures, e.g. of metal, such asstainless steel or copper. The vessel may have any useful shape, e.g. itmay be essentially cylindrical. The vessel may be associated withequipment for temperature control. The vessel may be used for preparingan aqueous extract of the finely divided cereal grains through a processinvolving incubation at one or more predefined temperatures as describedherein in the section “Preparing an aqueous extract”. Said equipment fortemperature control is capable of controlling the temperature of aliquid within the vessel, including being capable of heating a liquidwithin the vessel to a predetermined temperature, e.g. to any of thetemperatures described herein in the section “Preparing an aqueousextract”. The vessel (9) may also comprise equipment for stirring orrotating any liquid contained in said vessel. In particular, the vessel(9) may be a mashing vessel. Mashing vessels are well known in the art,and the vessel (9) may be any conventional mashing vessel.

The vessel (9), in general, contains an inlet (8), through which thefinely divided germinated cereal grains can enter the vessel. Said inlet(8) is typically positioned in the upper half of the vessel, e.g. in theupper ⅓, such as in the upper ⅕ of the vessel, for example at the top ofvessel. The finely divided cereal grains may be led via tubing from theequipment for finely dividing the cereal grains (7) to the inlet (8) ofthe vessel (9).

Generally, the vessel (9) also contains an outlet (10), through whichthe aqueous extract can exit the vessel after preparation of the aqueousextract (see details on the aqueous extract herein below in the sections“Aqueous extract” and “Preparing an aqueous extract”. The outlet istypically positioned in the lower half, for example in the lower ⅓, suchas in the lower ⅕ of the vessel, for example at the bottom of thevessel.

Finely Dividing Germinated Cereal Grains

The methods of the invention comprise a step of finely dividing cerealgrains germinated by incubation in an aqueous solution under aeration.

At the time that said cereal grains are finely divided they preferablystill have a high water content, preferably said cereal grains have awater content of at least 20%, more preferably of at least 25%, evenmore preferably of at least 30%, yet more preferably of at least 35%.For example, the germinated cereal grains may be transferred directlyfrom incubation in said aqueous solution to the equipment for finelydividing cereal grains. Accordingly, the germinated cereal grains mayhave the same water content at the time of being finely divided as thecereal grains have immediately after incubation of the cereal grains inthe aqueous solution, for example the water content described hereinabove in the section “Germination”. In particular, the methods do ingeneral not comprise a step of drying the germinated cereal grains bysubjection to elevated temperatures. Preferably, the germinated cerealgrains do not have a water content of less than 20%, preferably not lessthan 25%, even more preferably not less than 30%, yet more preferablynot less than 35% at any time after germination and prior to finelydividing said cereal grains. Thus, the methods do preferably notcomprise a step of kiln drying the germinated cereal grains.

The germinated cereal grains may be finely divided using any equipmentsuitable for finely dividing cereal grains having a water content ofmore than 20%, such as more than 25%, for example more than 30%, such asmore than 35%. For example, the germinated cereal grains may besubjected to milling, for example wet milling. Useful mills for millinggerminated cereal grains include the mills available from Millstar, USA.The germinated cereal grains may also be subjected to grinding.

The cereal grains are, generally, finely divided to an extent so that anaqueous extract of the fermentable sugars of the cereal grains can bemade. Thus, the cereal grains are sufficiently divided, such that a 7-Laqueous extract of 1 kg of said finely divided cereal grains has aspecific gravity of at least 8° Plato.

In embodiments of the invention, wherein the aqueous extract is madefrom the germinated cereal grains and one or more adjuncts, saidadjuncts may also be finely divided. In particular, this may be the casewhen said adjuncts comprise ungerminated cereal grains. Said adjunctsmay be finely divided, e.g. milled in separate proceedings. However, itis also comprised within the invention that the adjuncts are finelydivided together with the germinated cereal grains. Similarly, if theaqueous extract is made from the germinated cereal grains and kiln driedmalt, then said kiln dried malt may be finely divided, e.g. milled inseparate proceedings. However, it is also comprised within the inventionthat the kiln dried malt is finely divided together with the germinatedcereal grains.

Preparing an Aqueous Extract

The methods of the invention also comprise a step of preparing anaqueous extract of the finely divided germinated cereal grains. Saidstep may, for example, be a step of mashing.

The aforementioned aqueous extract may, in general, be prepared byincubating the finely divided cereal grains in water or in an aqueoussolution. The aqueous solution for preparing an aqueous extract is, ingeneral, a different aqueous solution as compared with the aqueoussolution used for incubation of the cereal grains during germination.

In order to distinguish, the aqueous solution for preparing an aqueousextract may also be referred to as “mashing solution”. The mashingsolution may be any aqueous solution, but it typically consists ofwater, such as tap water to which one or more additional agents may beadded. In order to distinguish between additional agents added duringgermination, these additional agents may be referred to as “additionalmashing agents”. Thus, the mashing solution may consist of water (e.g.tap water) to which one or more additional mashing agents are added. Themashing agents may be present in the mashing solution from the onset orthey may be added during the process of preparing an aqueous extract.

Said additional mashing agents may be enzymes. Thus, the mashingsolution may comprise one or more enzymes. Said enzymes may be added tothe mashing solution from the onset, or subsequently, during theprocess.

Said enzymes may, for example, be one or more hydrolytic enzymes.Suitable enzymes include lipases, starch degrading enzymes (e.g.amylases), glucanases [preferably (1-4)- and/or (1,3;1,4)-β-glucanases],and/or xylanases (such as arabinoxylanases), and/or proteases, or enzymemixtures comprising one or more of the aforementioned enzymes, e.g.Cereflo, Ultraflo, or Ondea Pro (Novozymes). For example, the mashingsolution may comprise one or more hydrolytic enzymes wherein at leastone hydrolytic enzyme is selected from the group consisting ofα-amylase, β-amylase, limit dextrinase, pullulanase, β-glucanase,xylanase, glucoamylase and protease.

In one embodiment of the invention the mashing solution comprises one ormore of the following enzymes:

-   -   A β-glucanase, such as an endo-(1,3;1,4)β-glucanase or an        endo-1,4-β-glucanase.    -   A xylanase, such as an endo- or exo-1,4-xylanase, an        arabinofuranosidase or a ferulic acid esterase    -   An α-amylase    -   A pullulanase or a limit dextrinase    -   A glucoamylase.

Whether or not to add enzymes to the mashing solution, and decisions onwhich enzymes to add, may dependent on the cereal grains used. Thus, inembodiments of the invention, wherein the cereal is a barley plant withlow levels of β-glucan (e.g. as described herein below in the section“Barley”), then little or no β-glucanase may be added to the mashingsolution.

In one embodiment it is preferred that no exogenous protease is addedduring mashing. Addition of protease may be less preferable, becauseproteases may affect enzyme activity. In one embodiment it is preferredthat no exogenous lipase is added during mashing.

In one embodiment it is preferred that at the most 700 U, preferably atthe most 350 U exogenous glucoamylase per g germinated cereal grains(dry matter) is used during preparation of the aqueous extract.

In one embodiment it is preferred that at the most 400 AGU, preferablyat the most 200 AGU exogenous glucoamylase per kg germinated cerealgrains (dry matter) is used during preparation of the aqueous extract.Determination of AGU may be performed as described in US7060468.

In another embodiment it is preferred that the combined exogenousglucoamylase and α-amylase used during preparation of the aqueousextract does not exceed 700 U, preferably does not exceed 350 U per ggerminated cereal grains (dry matter). The combined glucoamylase andα-amylase activity may for example be determined using K-CERA 01/12(protocol and kit available from Megazyme, Ireland).

In one embodiment it is preferred that at the most 20 U exogenouspullulanase or limited dextrinase per kg germinated cereal grains (drymatter) is used during preparation of the aqueous extract.

In one embodiment it is preferred that at the most 100 PUN pullulanaseper kg germinated cereal grains (dry matter) is used during preparationof the aqueous extract. Determination of PUN may be performed asdescribed in US7060468.

Said additional mashing agents may also be adjuncts, for exampleungerminated cereal grains, syrups or sugars. If adjuncts are added,these may also have been finely divided e.g. by milling or grinding. Ifthe adjunct is a cereal grain, for example a cereal grain, which has notbeen subjected to germination, then it may typically be finely dividedor milled. If the adjunct is syrups, sugars or the like, these willgenerally not be milled. Adjunct such as sugars or syrups may be addedto the mashing solution at any time in the process; however, suchadjuncts may also be added to the aqueous extract or later during theprocess for preparing a beverage as described below. In general, theadjuncts are added in smaller quantities than the germinated cerealgrains. Thus, at least 50%, preferably at least 70%, for example atleast 90% of the carbohydrates of the aqueous extract are derived fromthe germinated cereal grains, whereas adjuncts preferably only accountsfor a minor part of the carbohydrates. If the adjunct is an ungerminatedcereal grain, then it is preferred that the germinated cereal grainsconstitutes at least 50% (w/w), preferably at least 70% (w/w), morepreferably at least 90% (w/w) of the total cereal grains as determinedper dry weight.

The additional mashing agents may also be kiln dried malt. If kiln driedmalt is added, it may also have been finely divided e.g. by milling orgrinding. In general, the kiln dried malt is added in smaller quantitiesthan the germinated cereal grains. Thus, the germinated cereal grainsconstitute at least 80% (w/w), preferably at least 90% (w/w), morepreferably at least 95% (w/w) of the total cereal grains and malt asdetermined per dry weight. In preferred embodiments, no kiln dried maltis added.

Said additional mashing agents, preferably of food grade quality, mayalso be a salt, for example CaCl₂.

Said additional mashing agents may also be an acid, preferably a foodgrade acid, for example H₃PO₄.

The aqueous extract is generally prepared by incubation of the finelydivided germinated cereal grains in the mashing solution at one or morepredetermined temperature(s). Said predetermined temperature may also bereferred to as “mashing temperature” herein. Said mashing temperaturesmay for example be conventional temperatures used for mashing.

The mashing temperature is in general either kept constant (isothermalmashing), or gradually increased, for example increased in a sequentialmanner. In either case, soluble substances in the finely dividedgerminated cereal grains are liberated into said mashing solutionthereby forming an aqueous extract.

The mashing temperature(s) are typically temperature(s) in the range of30 to 90° C., such as in the range of 40 to 85° C., for example in therange of 50 to 85° C. The mashing temperatures may be chosen accordingto the cereal type used. Accordingly, in embodiments of the invention,wherein the cereal grains are barley with low levels of or absentlipoxygenase (LOX) activity and/or methyl methionine transferase (MMT)activity (see details herein below in the section “Barley”), the mashingtemperature may be lower, for example in the range of 35 to 69° C.

Incubation in the mashing solution may be performed for any suitableamount of time. The time for incubation in the mashing solution in themashing vessel may, e.g., be for in the range of 60 to 300 min, such asin the range of 60 to 240 min, for example in the range of 90 to 300min. such as in the range of 90 to 240 min, for example in the range of90 to 270 min. For example said time for incubation in the mashingsolution may be any time used in conventional mashing. One non-limitingexample of a suitable mashing is:

-   (1) Mashing-in at a temperature in the range of 50-60° C., such as    approximately 55° C., in the range of 10 to 30 min, such as    approximately 15 min.-   (2) Heating to a temperature in the range of 60 to 70° C.,    preferably in the range of 60 to 65° C., such as approximately 62°    C., in the range of 30 to 90 min, such as approximately 60 min.-   (3) Heating to a temperature in the range of 70 to 75° C., such as    approximately 72° C., in the range of 5 to 30 min, such as    approximately 15 min.-   (4) Heating to a temperature in the range of 75 to 80° C.,    preferably in the range of 75 to 78° C., such as approximately 78°    C., in the range of 5 to 15 min, such as approximately 10 min.

Subsequent to incubation in the mashing solution in the mashing vessel,the finely divided germinated cereal grains in the mashing solution maybe transferred to another container, e.g. a lauter tun and incubated foradditional time at elevated temperature, e.g. at in the range of 70 to78° C. for in the range of 30 to 120 min.

Thus, the incubation in the mashing solution may in addition toaforementioned steps also comprise a step (5) of:

-   (5) Heating to a temperature in the range of 70 to 78° C.,    preferably in the range of 75 to 78° C., such as approximately 78°    C., in the range of 30 to 120 min, such as approximately 60 min.

One non-limiting example of useful mashing temperatures and time isshown herein in FIG. 5 . The incubation for the first approximately 120min may for example be performed in a mashing vessel, whereas theremainder of the incubation for example may be performed in anothercontainer. Other non-limiting examples can be found in the literature ofbrewing, e.g. in Briggs et al. (supra) and Hough et al. (supra).

After incubation in the mashing solution, the aqueous extract maytypically be separated, e.g. through filtration into the aqueous extractand residual non-dissolved solid particles, the latter also denoted“spent grain”. Filtering may for example be performed in a lauter tun.Alternatively, the filtering may be filtering through a mash filter. Theaqueous extract thus obtained may also be denoted “first wort”.

Additional liquid, such as water may be added to the spent grains duringa process also denoted sparging. After sparging and filtration, a“second wort” may be obtained. Further worts may be prepared byrepeating the procedure.

Thus, the aqueous extract may be wort, e.g. a first wort, a second wort,a further wort or a combination thereof.

Aqueous Extract

The aqueous extract prepared by the methods of the invention may have anumber of useful properties, including—but not limited to—the propertiesdescribed in this section.

As mentioned above, the aqueous extract may be subjected to a step offiltration. Accordingly, it may be preferred that the wort has goodfilterability. For example, it may be technically challenging to filterhighly viscous liquid—a reason that it may be preferred that the aqueousextract has a low viscosity.

Filterability may be determined in a number of ways. In one embodimentthe filterability is determined as the amount of liquid obtained afterfiltration through a filter funnel equipped with a filter paper for 1 h.Preferably the aqueous extract has a filterability of at least 250 mL,when 400 mL mashing solution comprising 100 g finely divided cerealgrains is added to said filter funnel. Filterability may also bedetermined as the percentage of the volume of liquid obtained afterfiltration for 60 min as described above compared to the volume ofliquid of the aqueous extract added to said funnel. Thus, thefilterability may be at least 50%, such as of at least 60% (v/v). Inparticular, filterability may be determined as described herein below inExample 3.

Filterability may frequently be dependent on the level of β-glucan.Accordingly, it may be preferred that the level of β-glucan is not toohigh. For example the aqueous extract may comprise at the most 200 mg/L,preferably at the most 150 mg/L β-glucan.

It is also preferred that the aqueous extract comprises an adequatelevel of fermentable sugars. In particular it may be preferred that theaqueous extract comprises at least 10 g, such as at least 15 g maltoseper L. For example, it may be preferred that the aqueous extractcomprises at least 1 g/L per ° Plato maltose. It may also be preferredthat said aqueous extract comprises at least 1 g, such as at least 2 gglucose per L.

It is generally desirable that wort contains free amino nitrogen (FAN)at levels which are high enough to obtain good yeast viability, whereasvery high levels may be undesirable. Accordingly, it may be preferredthat the aqueous extract comprises in the range of 150 to 400 mg/L, suchas in the range of 150 to 300 mg/L, for example in the range of 150 to250 mg/L FAN.

It is generally desirable that wort contains high levels of the aminoacid valine, because that may reduce the likelihood of undesireddiacetyl formation. Accordingly, it may be preferred that the aqueousextract comprises at least 55 mg/L, for example at least 60 mg/L valine.In one embodiment, the aqueous extract comprises at least 65 mg/Lvaline.

Aforementioned levels of sugars, FAN and amino acids are preferablylevels in the aqueous extract prior to any fermentation.

Preparing Beverages

In some embodiments, the methods of the invention also comprise a stepof processing the aqueous extract prepared by the methods of theinvention into a beverage.

The aqueous extract may be boiled with or without hops where after itmay be referred to as boiled wort.

First, second and further worts may be combined, and thereaftersubjected to heating or boiling. The aqueous extract may be heated orboiled for any suitable amount of time, e.g. in the range of 60 min to120 min. During heating or boiling the volume of the aqueous extract maybe reduced due to evaporation. It may be preferred that the volume ofthe aqueous extract is reduced by less than 8%, preferably by less than5%. This may reduce energy consumption significantly.

The beverage may be prepared by fermentation of the aqueous extract,e.g. by fermentation of wort. Thus, the beverage may be prepared byfermentation of the aqueous extract with yeast.

In one embodiment, the beverage may be an alcoholic beverage, such asbeer. In other embodiments, the beverage may be a non-alcoholic beveragebased on germinated cereal grains. The non-alcoholic beverage, may forexample be a non-alcoholic beer or other kinds of non-alcoholicbeverages, such as maltina.

In one preferred embodiment the beverage is beer, for example the beermay be a lager beer or an ale. Thus, the beer may for example beselected from the group consisting of Altbier, Amber ale, Barley wine,Berliner weisse, Bière de Garde, Bitter, Blonde Ale, Bock, Brown ale,California Common, Cream Ale, Dortmunder Export, Doppelbock, Dunkel,Dunkelweizen, Eisbock, Fruit Iambic, Golden Ale, Gose, Gueuze,Hefeweizen, Helles, India pale ale, Kölsch, Lambic, Light ale, Maibock,Malt liquor, Mild, Märzenbier, Old ale, Oud bruin, Pale ale, Pilsener,Porter, Red ale, Roggenbier, Saison, Scotch ale, Steam beer, Stout,Schwarzbier, lager, Witbier, Weissbier and Weizenbock. The aqueousextract according to the invention is prepared from germinated cerealgrains, which have not been subject to kiln drying. Germinated cerealgrains, which have not been kiln dried, generally have a lighter colour,and accordingly, the methods of the invention are particularly usefulfor preparation of lighter beers, in particular for preparation of lagerbeer. Darker beers may also be prepared by the methods of the invention,e.g. by adding one or more kiln dried malts during mashing as describedin the section “Preparing beverages”.

Thus, the invention also relates to methods of producing a beveragecomprising the steps of:

-   -   Preparing an aqueous extract by the method according to the        invention.    -   Processing said extract into a beverage.

Alcoholic beverages—such as beer—may according to the methods of theinvention be manufactured from germinated cereal grains. Germinatedcereal grains, in addition to hops and yeast, contributes to flavour andcolour of the beer.

Once the aqueous extract has been prepared it may be processed into beerby any method including conventional brewing methods. Non-limiteddescriptions of examples of suitable methods for brewing can be found,for example, in publications by Briggs et al. (1981) and Hough et al.(1982). Numerous, regularly updated methods for analyses of barley andbeer products are available, for example, but not limited to, AmericanAssociation of Cereal Chemists (1995), American Society of BrewingChemists (1992), European Brewery Convention (1998), and Institute ofBrewing (1997). It is recognized that many specific procedures areemployed for a given brewery, with the most significant variationsrelating to local consumer preferences. Any such method of producingbeer may be used with the present invention.

The first step of producing beer from the aqueous extract preferablyinvolves heating said aqueous extract as described herein above,followed by a subsequent phase of cooling and optionally whirlpool rest.One or more additional compounds may be added to the aqueous extract,e.g. one or more of the additional compounds described below in thesection “Additional compounds”. After being cooled, the aqueous extractmay be transferred to fermentation tanks containing yeast, e.g. brewingyeast, such as S. pastorianus or S. cerevisiae. The aqueous extract maybe fermented for any suitable time period, in general in the range of 1to 20, such as 1 to 10 d. The fermentation is performed at any usefultemperature e.g. at a temperature in the range of 10 to 20° C. Themethods may also comprise addition of one or more enzymes, e.g. one ormore enzymes may be added to the wort prior to or during fermentation.In particular, said enzyme may be a proline-specific endoprotease. Anon-limiting examples of a proline-specific endoprotease is “Brewer'sClarex” available from DSM. In other embodiments, no exogenous enzymesare added during the methods.

During the several-day-long fermentation process, sugar is converted toalcohol and CO₂ concomitantly with the development of some flavoursubstances. The fermentation may be terminated at any desirable time,e.g. once no further drop in % P is observed.

Subsequently, the beer may be further processed, for example chilled. Itmay also be filtered and/or lagered—a process that develops a pleasantaroma and a less yeast-like flavour. Additives may also be added.Furthermore, CO₂ may be added. Finally, the beer may be pasteurizedand/or filtered, before it is packaged (e.g. transferred to containersor kegs, bottled or canned). The beer may also be pasteurized bystandard methods.

The beer produced by the methods of the invention typically have apleasant taste, and lacks or only have little astringency. Taste may beanalyzed, for example, by a specialist beer taste panel.

Barley

In preferred embodiments of the invention the cereal grains to be usedwith the methods of the invention are barley grains.

Said grains may be grains of any barley plant. However, in someembodiments, the barley plant may comprise one or more specificcharacteristics, for example, one or more of the characteristics asdescribed herein below. Even though the various characteristics arediscussed individually herein below, the barley plant of the inventionmay have a combination of these characteristics.

In one embodiment of the invention, the barley may be a hull-less barleyvariety (var.). It is also comprised within the invention that thebarley is a barley var. with naturally thin husk, such as var. Admiral.For example, the husk may constitute less than 7% of the total weight ofgrain and husk.

As mentioned above, it is preferable that the aqueous extract obtainedduring mashing has a viscosity sufficiently low to allow goodfilterability of the mash mixture. As also described in detail above,soluble β-glucans may contribute to high viscosity of an aqueousextract. Accordingly, in some embodiments of the invention, it may bepreferred to use a cereal plant—and in particular a barley plant havinga low level of β-glucan, e.g. no β-glucan, such as a level of β-glucanthat is below the detection level. These barley plants are known in theart and include, for example, barley plants carrying a mutation in thegene encoding a β-glucan synthase. Said gene may be a gene encoding thepolypeptide of SEQ ID NO:2 set forth in US2012/0030784. For example, thebarley plant may be a barley comprising a β-glucan-deficient gene as setforth in SEQ ID NO:1 or SEQ ID NO:18 of US2012/0030784. The barley plantmay also be that containing a silenced CsIF6 gene, leading to barleygrains with very low levels of (1,3;1,4)-β-glucan (as described byTaketa et al., 2011).

The barley plant may also be a barley plant having a low level of LOXactivity. Such barley plants are known in the art, and include, forexample, barley plants carrying a mutation in the gene encoding LOX-1.For example, the barley plant may be a barley plant carrying any of themutations in the LOX-1 gene described in WO 02/053721, WO 2005/087934and WO 2004/085652.

The barley plant may also be a barley plant carrying a mutation in thegene encoding lipoxygenase 1 (LOX-1) and/or in the gene encoding LOX-2.For example, the barley plant may be a barley plant carrying any of themutations in the LOX-1 and LOX-2 genes described in WO 2010/075860.

The barley plant may also be a barley plant having a low level of MMTactivity. Such barley plants are known in the art and include, forexample, barley plants carrying a mutation in the gene encoding MMT.Specifically, the barley plant may be a barley plant carrying any of themutations in the MMT gene described in WO 2010/063288. The barley plantmay also be any of the barley plants described in WO 2011/150933.

The barley plant may also be a barley plant characterised by increasedGA signalling. In particular, the barley plant may be a barley plantcarrying a mutation in the Slender1 gene, which encodes the DELLAprotein. For example, the barley plant may be a barley plant carryingany of the mutations described by Chandler et al., Journal ofExperimental Botany, Vol. 64, No. 6, pp. 1603-1613, 2013,doi:10.1093/jxb/ert022, e.g. in Table 1 therein. For example, the barleyplant may carry a mutation in the Slender1 gene resulting in a mutantSlender1 gene encoding a mutant DELLA protein, wherein said mutant DELLAprotein carries a mutation in one or more of amino acids number 46, 490,280, 268, 271, 277, 231, 481, 282, 277, 227, 485 or 237, for example amutation selected from the group consisting of G46E, S490F, R268H,G271D, A277T, V231M, R481H, V282F, A277T, G227E, S485F and C237Y. Theamino acid numbering is provided in relation to the sequence of theDELLA protein available under the Genbank accession no. AK372064 orAF035820 (version as of 4 Feb. 2013).

Beverage

Beverages prepared by processing an aqueous extract according to theinvention into a beverage may have a number of useful properties,including—but not limited to—the properties described in this section.

It is generally desirable that the beverages according to the inventioncontain as little diacetyl as possible. Accordingly, it may be preferredthat the beverage comprises diacetyl at a level, which is below thethreshold considered off-flavor in lager beer. Preferably, the beveragecomprises at the most 30 ppb diactyl, more preferably at the most 25 ppbdiacetyl, even more preferably at the most 20 ppb diacetyl. This is inparticular the case if the beverage is beer, for example lager beer.

The beverage according to the present invention may for example be anaqueous extract as described herein, which optionally has beenfermented. Thus, the beverage may comprise or consist of said aqueousextract or fermented aqueous extract and optionally one or moreadditional compounds. Said additional compounds may for example be anyof the additional compounds described herein below in the section“Additional compounds”.

Additional Compounds

The methods of the invention may comprise the step of adding one or moreadditional compounds. Said additional compounds may for example be aflavor compound, a preservative, a functional ingredient, a color, asweetener, a pH regulating agent or a salt. The pH regulating agent mayfor example be a buffer or an acid, such as phosphoric acid.

Functional ingredients may be any ingredient added to obtain a givenfunction. Preferably a functional ingredient renders the beveragehealthier. Non-limiting examples of functional ingredients includesvitamins or minerals.

The preservative may be any food grade preservative, for example it maybe benzoic acid, sorbic acid, sorbates (e.g. potassium sorbate),sulphites and/or salts thereof.

The additional compound may also be CO₂. In particular, CO₂ may be addedto obtain a carbonated beverage.

The flavour compound to be used with the present invention may be anyuseful flavour compound. The flavour compound may for example beselected from the group consisting of aromas, plant extracts, plantconcentrates, plant parts and herbal infusions. In particular the flavorcompounds may be hops.

Items

The invention may further be described by the following items:

-   1. A method for producing an aqueous extract of a cereal, said    method comprising the steps of:    -   a. providing grains of a cereal;    -   b. incubating said grains in an aqueous solution until the        grains have a water content of at least 35%, wherein at least 2        L O₂ per kg dry weight cereal grains is passed through said        aqueous solution per h, thereby producing germinated grains;    -   c. finely dividing said germinated grains, while said germinated        grains have a water content of at least 35%;    -   d. preparing an aqueous extract of said milled germinated        grains,    -   thereby producing an aqueous extract of the cereal.-   2. A method for producing an aqueous extract of a cereal, said    method comprising the steps of:    -   a. providing grains of a cereal;    -   b. subjecting the cereal grains to a step of germination thereby        obtaining germinated grains, wherein said step of germination        comprises incubating said grains in an aqueous solution until        the grains have a water content of at least 30%, wherein at        least 2 L O₂ per kg dry weight cereal grains is passed through        said aqueous solution per h;    -   c. finely dividing said germinated grains, while said germinated        grains have a water content of at least 20%; with the proviso        that said cereal grains do not have a water content below 20% at        any time between steps b) and c),    -   d. preparing an aqueous extract of said milled germinated        grains,    -   thereby producing an aqueous extract of the cereal.-   3. The method according to any one of the preceding items, wherein    said grains are incubated in said aqueous solution for in the range    of 20 to 72 h.-   4. The method according to any one of the preceding items, wherein    the grains of the cereal are submerged in the aqueous solution    during the entire step of germination.-   5. The method according to any one of the preceding items, wherein    the grains of the cereal are submerged in aqueous solution, wherein    at least 2 L O₂ per kg dry weight cereal grains is passed through    said aqueous solution per h for in the range of 24 to 60 h,    preferably for in the range of 40 to 55 h.-   6. The method according to any one of items 1 to 3, wherein the step    of germination comprises    -   i. at least one step of incubating said grains in an aqueous        solution, wherein at least 2 L O₂ per kg dry weight cereal        grains is passed through said aqueous solution per h; and    -   ii. at least one step of incubating said cereal grains in air.-   7. The method according to item 6, wherein said at least one step of    incubating said grains in an aqueous solution is performed for in    the range of 16 to 40 h, preferably for in the range of 20 to 30 h.-   8. The method according to any one of items 6 to 7, wherein the step    of incubating said cereal grains in air is performed under aeration.-   9. The method according to any one of items 6 to 8, wherein the step    of incubating said cereal grains in air is performed for in the    range of 18 to 50 h, more preferably for in the range of 18 to 38 h,    for example for in the range of 22 to 35 h.-   10. The method according to any one of the preceding items, wherein    the entire step of germination does not exceed 72 h, more preferably    does not exceed 60 h, even more preferably does not exceed 54 h.-   11. The method according to any one of the preceding items, wherein    said aqueous solution is water.-   12. The method according to any one of the preceding items, wherein    the method further comprises adding gibberellic acid (GA) to the    aqueous solution.-   13. The method according to item 12, wherein said GA is added to the    aqueous solution at a concentration of at least 100 nM, for example    at least 1000 nM.-   14. The method according to any one of the preceding items, wherein    at least 3 L, more preferably at least 4 L, yet more preferably at    least 5 L, even more preferably at least 6 L O₂ per kg dry weight of    cereal grains is passed through said aqueous solution per h.-   15. The method according to any one of the preceding items, wherein    at least 20 g O₂ per kg cereal grain, more preferably at least 30 g    O₂ per kg cereal grain, yet more preferably at least 40 g O₂ per kg    cereal grain, for example in the range of 40 to 100 g O₂ per kg    cereal grain, such as in the range of 40 to 80 g O₂ per kg cereal    grain, for example in the range of 60 g O₂ per kg cereal grain (dry    matter) is passed through said aqueous solution/cereal grain mixture    per h.-   16. The method according to any one of the preceding items, wherein    said O₂ is comprised within a gas mixture.-   17. The method according to item 16, wherein the gas mixture is    atmospheric air.-   18. The method according to any one of the preceding items, wherein    at least 10 L, preferably at least 15 L, more preferably at least 20    L, yet more preferably at least 25 L, even more preferably at least    30 L atmospheric air per kg dry weight cereal grains is passed    through said aqueous solution per h.-   19. The method according to any one of the preceding items, wherein    the aqueous solution further comprises an antifoaming agent.-   20. The method according to any one of the preceding items, wherein    the incubation is performed at a temperature in the range of 15 to    30° C., preferably at approximately 25° C.-   21. The method according to any one of the preceding items, wherein    the grains are kept in the same vessel during step b.-   22. The method according to any one of the preceding items, wherein    the cereal grains are incubated in said aqueous solution until they    have a water content of at least 35%, such as at least 37%.-   23. The method according to any one of the preceding items, wherein    the grains provided in step a., have been treated with an    antimicrobial agent.-   24. The method according to item 23, wherein the antimicrobial agent    is a peroxide, such as hydrogen peroxide.-   25. The method according to any one of the preceding items wherein    the method does not comprise a step of rootlet removal.-   26. The method according to any one of the preceding items, wherein    the cereal is a hulled cereal, for example a hulled barley.-   27. The method according to item 26, wherein the method comprises a    step of removing at least part of said hull prior to incubating said    grains in an aqueous solution.-   28. The method according to item 27, wherein removal of said hull    results in a loss of in the range of 1 to 4%, such as in a loss of    in the range of 1.5 to 3.0% of the total weight of the cereal    grains.-   29. A method for producing an aqueous extract of a cereal, said    method comprising the steps of:    -   A. providing germinated grains of a cereal having a water        content of at least 35%;    -   B. finely dividing said germinated grains, while said germinated        grains have a water content of at least 35%;    -   C. preparing an aqueous extract of said milled germinated        grains,    -   thereby producing an aqueous extract of the cereal.-   30. A method for producing an aqueous extract of a cereal, said    method comprising the steps of:    -   A. providing germinated grains of a cereal having a water        content of at least 20% with the proviso that said cereal grains        have not had a water content below 20% at any time subsequent to        germination;    -   B. finely dividing said germinated grains, while said germinated        grains have a water content of at least 20%;    -   C. preparing an aqueous extract of said milled germinated        grains,    -   thereby producing an aqueous extract of the cereal.-   31. The method according to any one of the preceding items, wherein    the germinated cereal grains have a water content of at least 25%,    even more preferably of at least 30%, yet more preferably of at    least 35% at the time of finely dividing said cereal grains.-   32. The method according to any one of the preceding items, wherein    the germinated cereal grains have not had a water content of less    than 25%, even more preferably of less than 30%, yet more preferably    of less than 35% at any time between completion of the step of    germination and the time of finely dividing said cereal grains.-   33. The method according to any one of the preceding items, wherein    the cereal is barley.-   34. The method according to item 33, wherein the barley is a    hull-less barley or a barley variety having a thin husk.-   35. The method according to any one of items 1 to 32, wherein the    cereal is a hull-less cereal, for example wheat or a hull-less    barley.-   36. The method according to any one of items 1 to 33, wherein the    cereal is a hulled cereal, such as a hulled barley.-   37. The method according to any one of the preceding items, wherein    the cereal is a barley characterized by one or more of the    following:    -   A. Carrying a mutation in the gene encoding a β-glucan synthase    -   B. Carrying a mutation in the gene encoding LOX-1    -   C. Carrying a mutation in the gene encoding LOX-2    -   D. Carrying a mutation in the gene encoding MMT; and/or    -   E. Carrying a mutation in the gene encoding DELLA-   38. The method according to any one of the preceding items, wherein    the germinated grains have an α-amylase activity of at least 4 U/g,    preferably at least 30 U/g cereal grain on a dry weight basis.-   39. The method according to any one of the preceding items, wherein    the germinated grains have an α-amylase activity of at least 100 U/g    cereal grain on a dry weight basis.-   40. The method according to any one of the preceding items, wherein    the germinated grains have a β-amylase activity of at least 5 U/g    cereal grain on a dry weight basis.-   41. The method according to any one of the preceding items, wherein    the germinated grains have a limit dextrinase activity of at least 5    mU/g grain on a dry weight basis.-   42. The method according to any one of the preceding items, wherein    the germinated grains contains at the most 4 g rootlets (dry matter)    per 100 g germinated cereal grains (dry matter).-   43. The method according to any one of the preceding items, wherein    the germinated grains contains at the most 2 g rootlets (dry matter)    per 100 g germinated cereal grains (dry matter).-   44. The method according to any one of the preceding items, wherein    the germinated grains immediately prior to the step of finely    dividing them have a content of nitroamines of at the most 0.15    μg/kg, preferably at the most 0.12 μg/kg, for example at the most    0.10 μg/kg cereal grains (dry matter).-   45. The method according to any one of the preceding items, wherein    step C. comprises mashing said milled germinated grains with a    mashing solution at a temperature in the range of 50 to 80° C.-   46. The method according to item 45, wherein said mashing is    performed in the presence of one or more added hydrolytic enzyme(s).-   47. The method according to item 46, wherein at least one hydrolytic    enzyme is selected from the group consisting of cell wall- and    starch-degrading enzymes, including, but not limited to, α-amylase,    β-amylase, limit dextrinase, pullulanase, β-glucanase, xylanase,    glucoamylase and protease.-   48. The method according to item 45, wherein said mashing is    performed in the presence of at least one β-glucanase and at least    one xylanase.-   49. The method according to any one of items 45 to 48, wherein at    the most 700 U, preferably at the most 350 U exogenous glucoamylase    and/or α-amylase per g germinated cereal grains (dry weight) is    added during said mashing.-   50. The method according to any one of items 45 to 49, wherein at    the most 100 PUN exogenous pullulanase per g germinated cereal    grains (dry weight) is added during said mashing.-   51. The method according to any one of items 45 to 46 and 49 to 50,    wherein the cereal is characterized by a low β-glucan level in the    grains, and wherein no β-glucanase is added during mashing.-   52. The method according to any one of the preceding items, wherein    the method further comprises a step of filtering said aqueous    extract.-   53. The method according to any one of the preceding items, wherein    the aqueous extract has a filterability of at least 50%, such as of    at least 60%.-   54. The method according to any one of the preceding items, wherein    the aqueous extract comprises at the most 200 mg/L β-glucan.-   55. The method according to any one of the preceding items, wherein    the aqueous extract comprises at least 10 g, such as at least 15 g    maltose per L.-   56. The method according to any one of the preceding items, wherein    the aqueous extract comprises in the range of 150 to 400 mg/L FAN.-   57. The method according to any one of the preceding items, wherein    the aqueous extract comprises at least 60 mg/L, preferably at least    65 mg/L valine.-   58. The method according to any one of the preceding items, wherein    the method does not comprise a step of kiln drying.-   59. A method for producing a beverage, said method comprising the    steps of:    -   i. preparing an aqueous extract by the method according to any        one of the preceding items;    -   ii. processing said extract into a beverage.-   60. The method according to item 59, wherein step ii. comprises the    steps of:    -   a. heating said aqueous extract optionally in the presence of        hops or hops extract;    -   b. cooling the aqueous extract;    -   c. fermenting said aqueous extract with yeast, thereby producing        a fermented beverage.-   61. The method according to item 60, wherein the method further    comprises a step of sedimentation performed after step a. or step b.-   62. The method according to any one of of the preceding items,    wherein the entire method is performed at one site.-   63. The method according to any one of items 59 to 62, wherein the    beverage comprises at the most 25, such as at the most 20 ppb    diacetyl.

EXAMPLES

The invention is further illustrated by the following examples. These,however, should not be considered as limiting for the invention. Thebarley samples used in the examples herein below were all analysed asfollows:

Germination Test

-   -   All barley samples used in the examples were evaluated for the        parameters germination index, germination energy and water        sensitivity. Data was based on a sample size of 100 barley        grains for a 4-mL germination test and a sample size of 100        barley grains for a 8-mL germination test according to        Analytica-EBC Method 3.6.2 Germinative Energy of Barley (BRF        Method).

Characterization of Barley Samples

-   -   Thousand kernel weights were determined by automatic counting        using a Data Count JR instrument, while size fractionation        utilized a Pfeuffer Sortimat K3 adjusted to 4 classes of        different grain with (X): X>2.8 mm; 2.8<X>2.5 mm; 2.5<X>2.2 mm;        X<2.2 mm. Size fractionation data was calculated based on 100-g        grain samples.    -   Protein, water and starch contents of barley samples were        determined using a Foss 1241 NIT instrument, using barley        calibration (FOSS BY213271; provided by Foss, D K). Prior (e.g.        24 h) to incubation in aqueous solution, the water content of        the 100-g grain samples were re-determined using a Foss 1241 NIT        instrument using barley calibration Foss BY303300 (Foss,        Denmark).    -   The water content of grain was determined by first measuring the        weight of the corresponding barley sample, followed by drying        said sample and determining the weight of the dried sample. The        difference in weight of the wet and dry sample is considered to        be water, and the water content equals the weight of the water        divided by the total weight of the sample (wet sample).

Analysis of Germinated Grains

-   -   Samples of germinated grains were tested for the following        parameters (w/w): water content, protein content, soluble        protein and extract of the malt sample. Values were determined        using a Foss 1241 NIT instrument calibrated according to data        provided by Foss (DK; calibration MA000010).

Example 1. Single-Step Steeping and Germination

In the laboratory-scale experiments, 1 kg of dry barley grain was placedin a Plexiglass cylinder and constantly aerated with atmospheric airfrom beneath the column of grain. A schematic drawing of the equipmentused is provided herein in FIG. 8 . The grain was aerated from beneathwith varying levels of atmospheric air for different time period, duringwhich the grain moisture content raised as shown in Table 1 andgermination is initiated. Different barley varieties, as indicated inthe figure legend were used in this analysis. Airflow was set using aSmartTrak® 50 mass flow meter and controller (Sierra, Calif., USA) andtemperature was measured using a Testo 735 precision thermometer (Testo,Germany).

Sensors for measuring airflow, temperature, pH, conductivity, redoxpotential and O₂ content of the steeping water were incorporated in thissystem. The sensors allow not only the process to be monitored in realtime, but also to adjust steeping and germination conditions during theprocesses; this level of control is not possible by following currentmalting and brewing protocols.

Grains of the a hull-less barley line were transferred to the Plexiglascylinder and first incubated for 3 h in 1% P3-hypochloran (Ecolab,Switzerland) following incubation for 45 h in water adjusted to 1 nMgibberellic acid (GA) and 0.01% Foamazol FCD511 (AB Vickers, Burton onTrent, UK). Incubation was at either 15 or 25° C., and the grains wereaerated with either 30, 60, 90 or 120 L/h atmospheric air. Samples werecollected after 24 h and 48 h. The results are summarised in FIGS.2A-2B. As shown air access strongly promoted barley germination. Whencomparing to the non-aerated sample (0 L/hr) all samples subjected to anairflow were characterised by a notable difference in grain development.In particular, the grains had a visible chit of more than 1 mm evenafter 24 h, at 15° C. and 30 L/h airflow. Increasing the airflow causedadditional chit development after 24 h at 15° C. At 25° C. some grainseven developed visible rootlets (60, 90 or 120 L/h). Increasing theincubation time lead to advance in development, with all grainssubjected to airflow characterised by germination and development ofvisible rootlets after 48 h. With increased incubation temperature,there was enhanced development of chits and rootlets. An airflow of 90L/h corresponds to 51 g O₂ per h. If calculated as O₂ per L H₂O, theamount will vary over time, because the cereal grains take up waterduring incubation. Typically, an airflow of 90 L/h corresponds to 64-121g O₂ per L H₂O per h.

The same experiment was performed using grains of a hulled barley lineand the results are summarised in FIGS. 3A-3B. Grains of the hulledbarley line also had a visible chit of more than 1 mm after incubationat 24 h, at 25° C. and 30 L/h airflow. Increasing the incubation timelead to advance in development with all grains subjected to airflow of60 L/h characterised by germination and development of visible rootletsafter 48 h.

The water uptake in grains was asserted by determining the water contentas % (w/w) in a hull-less barley line and in a hulled barley line, at15° C. and 25° C. as described above. The results are summarised inTable 1 (hull-less barley) and Table 2 (hulled barley) below. The watercontent seems not to be highly dependent on the airflow, if the airflowis at least 30 L/h. In contrast, the water content was much higher after24 h at 25° C. than at 15° C.

TABLE 1 Water uptake (%), hull-less barley grains Air Temp. (T) = 15° C.T = 25° C. flow 0 h 24 h 48 h 0 h 24 h 48 h  0 L/h 11.4 31.7 ± 0.6 37.1± 1.4 11.4 36.7 ± 1.3 43.3 ± 0.9  30 L/h 11.4 35.5 ± 1.5 41.5 ± 1.8 11.440.6 ± 0.5 46.0 ± 0.1  60 L/h 11.4 35.1 ± 1.0 42.0 ± 1.2 11.4 38.9 ± 0.146.5 ± 0.6  90 L/h 11.4 34.3 ± 0.9 42.6 ± 1.4 11.4 38.3 ± 1.7 45.7 ± 0.5120 L/h 11.4 35.5 ± 0.8 43.2 ± 1.1 11.4 38.2 ± 2.2 44.9 ± 0.6

TABLE 2 Water uptake (%), hulled barley grains Air Temp. (T) = 15° C. T= 25° C. flow 0 h 24 h 48 h 0 h 24 h 48 h  0 L/h 13.6 32.5 ± 0.7 36.5 ±0.3 13.6 38.3 ± 0.6 42.6 ± 0.7  30 L/h 13.6 33.4 ± 0.3 39.0 ± 0.4 13.639.3 ± 0.2 46.7 ± 0.6  60 L/h 13.6 32.9 ± 0.7 39.4 ± 0.6 13.6 39.7 ± 1.247.9 ± 0.2  90 L/h 13.6 34.1 ± 0.3 38.5 ± 0.2 13.6 40.3 ± 0.4 47.2 ± 0.5120 L/h 13.6 33.3 ± 0.5 39.0 ± 0.4 13.6 40.1 ± 0.3 47.3 ± 0.8

Accordingly, results of the present invention show that a temperature of25° C. may be more preferable for early rates of water uptake by thegrain and hence in the overall speed of the germination.

Example 2. Enzyme Activity

During germination, the barley grain begins to secrete a range ofhydrolytic enzymes, such as α-amylases, limit dextrinases and(1,3;1,4)-β-glucanases. Typically, these enzyme activities are detectedin a timely coordinated manner, with the activities of α-amylase,β-amylase and/or limit dextrinase useful as a general marker foractivity of hydrolytic enzymes. Thus, the activities of α-amylase andlimit dextrinase were determined after germination performed accordingto the method of the invention.

GA is a phytohormone that activates the aleurone layer in germinatingbarley. Many maltsters add GA at low concentration during the maltingprocess. Here, various concentrations of GA supplemented to the waterfor incubation of the grains at the start of the process. A GA₃ solutionwas prepared from gibberellic acid (G7645, Sigma-Aldrich, St. Louis,Mo., USA) in absolute ethanol and added to the water. The enzymeactivities were monitored by measuring hydrolytic enzyme activities ingrain extracts at 24 hr and 48 hr.

Sample Preparation

Prior to enzyme activity analysis the germinated grain samples weremilled using a standard Foss Cyclotech mill (Foss, Denmark), equippedwith a tungsten carbide grinding ring (Foss 10004463), nickel platedimpeller (Foss 1000 2666) and a 1 mm outlet screen (Foss 10001989). Allmeasurements of enzyme activity in germinated barley grains were madewithin 48 h after milling of the sample.

α-Amylase Activity

α-Amylase activity of germinated grains was based on flour prepared asdescribed above in the section “Sample preparation”. Assays fordetermination of α-amylase activity utilized a Ceralpha kit kit fromMegazyme using standard laboratory equipment. The assays were madeaccording to manufacturer's protocol (K-CERA 01/12), includingcalculation of α-amylase activity.

β-Amylase Activity

When measuring beta-amylase activity of germinated grains, flour wasmade as described above in the section “Sample preparation”. 3-Amylaseactivity assays followed the recommendations provided with the theBetamyl kit from Megazyme (K-BETA3).

Limit Dextrinase Activity:

For measurement of limit dextrinase activity in germinated grains, flourwas made as described above in the section “Sample preparation”. Limitdextrinase activity was determined using a Limit Dextrizyme kitT-LDZ1000 from Megazyme. Assays, including activity measurements, weredone according to manufacturer's protocol (T-LDZ1000 07/9).

α-Amylase, β-amylase and limit dextrinase activities were determined ina hull-less barley line. The barley grains were germinated essentiallyas described in Example 1 by incubation in water in the presence of0.01% FCD511 Foamzol and varying amounts of GA under aeration. Airflowwas set to 90 L/h using a SmartTrak® 50 mass flow meter and controller(Sierra, Calif., USA) without sanitation at 25° C. (measured using aTesto 735 precision thermometer, Testo, Germany). The enzyme activitiesin the germinated grains were measured at 24 and 48 h after imbition,with the results shown in FIGS. 4A-4C.

In addition, α-amylase, β-amylase and limit dextrinase activities weredetermined in both a hull-less barley line and a hulled barley line. Thebarley grains were germinated essentially as described in Example 1 byincubation in water in the presence of 0.01% FCD511 Foamzol and 1000 nMGA under aeration. Varying airflow was used as indicated in Tables 3 and4 below using a SmartTrak® 50 mass flow meter and controller (Sierra,Calif., USA) without sanitation at 25° C. (measured using a Testo 735precision thermometer, Testo, Germany). The enzyme activities in thegerminated grains were measured at 24 and 48 h, with the results shownin Table 3 (hull-less barley) and Table 4 (hulled barley).

TABLE 3 Enzyme activities in grains of hull-less barley. Temperature (T)= 15° C. T = 25° C. Air flow 0 h 24 h 48 h 0 h 24 h 48 h  0 L/h α*) 1.1± 0.2 0.2 ± 0.2 n.d. 1.1 ± 0.2 n.d. n.d. β**) 18.6 ± 0.3  19.4 ± 0.1 16.2 ± 2.6  18.6 ± 0.3  15.8 ± 0.6  16.8 ± 1.6  LD***) 1.6 ± 2.2 0.3 ±0.4 0.4 ± 0.9 1.6 ± 2.2 1.1 ± 1.6 1.4 ± 0.8  30 L/h α 1.1 ± 0.2 2.0 ±0.2 13.6 ± 0.1  1.1 ± 0.2 5.2 ± 0.7 33.1 ± 0.6  β 18.6 ± 0.3  13.8 ±0.3  18.3 ± 1.8  18.6 ± 0.3  16.3 ± 0.8  19.6 ± 0.6  LD 1.6 ± 2.2 4.6 ±3.3 5.5 ± 0.9 1.6 ± 2.2 5.2 ± 0.6 14.0 ± 3.1   60 L/h α 1.1 ± 0.2 0.6 ±0.1 13.4 ± 0.8  1.1 ± 0.2 5.2 ± 0.1 48.3 ± 1.6  β 18.6 ± 0.3  15.0 ±1.8  18.6 ± 2.1  18.6 ± 0.3  15.5 ± 2.0  16.9 ± 3.7  LD 1.6 ± 2.2 3.6 ±0.2 7.5 ± 2.9 1.6 ± 2.2 4.8 ± 1.9 19.6 ± 2.5   90 L/h α 1.1 ± 0.2 0.6 ±0.1 13.8 ± 0.3  1.1 ± 0.2 5.2 ± 0.1 59.3 ± 2.3  β 18.6 ± 0.3  16.7 ±1.6  16.9 ± 1.4  18.6 ± 0.3  15.1 ± 3.6  17.7 ± 2.5  LD 1.6 ± 2.2 1.5 ±1.4 7.5 ± 0.6 1.6 ± 2.2 4.0 ± 1.6 26.0 ± 3.3  120 L/h α 1.1 ± 0.2 0.6 ±0.1 12.7 ± 0.7  1.1 ± 0.2 4.9 ± 0.1 59.3 ± 0.9  β 18.6 ± 0.3  16.9 ±1.8  17.7 ± 1.7  18.6 ± 0.3  14.6 ± 3.1  18.6 ± 3.4  LD 1.6 ± 2.2 2.1 ±1.0 7.6 ± 0.4 1.6 ± 2.2 2.0 ± 0.4 22.4 ± 0.7  *)α-Amylase activity(U/g); **)β-amylase activity (U/g); ***)activity of free limitdextrinase (mU/g)

TABLE 4 Enzyme activities in grains of hulled barley *). Temperature (T)= 15° C. T = 25° C. Air flow 0 h 24 h 48 h 0 h 24 h 48 h  0 L/h α n.d.n.d. n.d. n.d. n.d. n.d. β 17.1 ± 0.7  19.2 ± 1.7  18.2 ± 1.0  17.1 ±0.7  12.8 ± 1.4  10.7 ± 0.5  LD n.d. 2.7 ± 2.6 n.d. n.d. 1.6 ± 2.2 1.6 ±0.2  30 L/h α n.d. n.d. n.d. n.d. n.d. 1.9 ± 2.7 β 17.1 ± 0.7  17.3 ±1.7   11 ± 2.7 17.1 ± 0.7  12.1 ± 1.4  14.5 ± 0.4  LD n.d. 2.7 ± 0.6n.d. n.d. 0.3 ± 0.4 2.1 ± 0.6  60 L/h α n.d. n.d. n.d. n.d. n.d. 4.4 ±6.2 β 17.1 ± 0.7  18.9 ± 0.5  11.0 ± 0.7  17.1 ± 0.7  9.8 ± 0.9 17.5 ±1.2  LD n.d. 0.6 ± 0.8 n.d. n.d.   0 ± 1.8 5.1 ± 0.4  90 L/h α n.d. n.d.n.d. n.d. n.d 4.1 ± 5.8 β 17.1 ± 0.7  18.6 ± 1.0  10.3 ± 1.0  17.1 ±0.7  9.3 ± 0.6 17.2 ± 1.2  LD n.d. n.d. n.d. n.d.   0 ± 1.4 9.2 ± 2.6120 L/h α n.d. n.d. n.d. n.d. n.d 9.3 ± 0.2 β 17.1 ± 0.7  18.6 ± 0.6 10.9 ± 1.3  17.1 ± 0.7  9.2 ± 0.6 16.0 ± 1.2  LD n.d. n.d. n.d. n.d. 2.4± 0.6 8.0 ± 2.2 *) Abbreviations are identical to those of the legend toTable 3; n.d. = not determined.

As illustrated in FIGS. 4A-4C, levels of both α-amylase and limitdextrinase increased significantly following addition of GA.

Example 3. Mashing

Barley grains were germinated as described herein above in Example 1.After 2 d of continuous steeping and germination, the liquid phase wasdrained from the grains and the grain was wet-milled in alaboratory-scale homogenizer (Omega Juicer 8226, Omega, USA). Extractionof the milled grains in water was done using the mashing scheduleoutlined in FIG. 5 . This process may also be referred to as “mashing”.During mashing a saccharification in general also occurs. During mashingCaCl₂ and H₃PO₄ is typically added to the water.

During the process of industrial mashing, exogenous enzyme preparationsmay be added to continue the conversion of partially degraded starch,storage protein and cell wall polysaccharides to fermentable sugars andamino acid that subsequently support the growth of the yeast duringfermentation. Mashing characteristics in the presence and absence of theUltraflo Max brewing enzyme mixture (Novozymes, Denmark) were compared.Ultraflo Max is an enzyme mixture comprising β-glucanase and xylanaseactivities. Following mashing, extracts were filtered using a standardmash filter.

The efficacy of the exogenous enzyme mixture was tested by measuring thefilterability of the mash mixture that remained after the mashingprocess. The filterability was determined using a 140-mm TOP ID filterfunnel (Urbanti Pequannock, N.J. USA) equipped with MN 614 ¼ Ø 320 mmREF 527032 (Macherey Nagel Duren Germany). The weight of samples wererecorded using a standard balance (MPB1502 L, Mettler Toledo,Switzerland). The filterability was determined as the total amount ofliquid obtained after filtration for 60 min of a mash mixture comprising400 mL of mashing solution, previously supplemented with 100 g ofmilled, germinated barley.

The experimental results are summarized in FIG. 6 for grains of ahull-less barley line, which was germinated for 48 h at 25° C. withaeration as that described for the experiment detailed in Example 1.

Example 4. Wort

Wort was prepared as detailed in the experiments of Example 3, usinggerminated barely grains prepared as described in Example 1. The barleygrains of a hull-less line were incubated for 48 h at 25° C. in thepresence of GA, as described in Example 1, under aeration withatmospheric air at either 45 L/h or 90 L/h. The germinated barley grainswere wet-milled and mashed as described in Example 3—either in thepresence or absence of the Ultraflo Max enzyme mixture (Novozymes,Denmark).

The levels of fermentable sugars—fructose, sucrose, glucose, maltose andmaltotriose—were determined in the following manner. After boiling ofthe wort, it was diluted 1:2000 with milliQ-water and subsequentlyfiltered through a 0.2-μm nylon membrane filter (Titan3 30 mm, ThermoScientific, CA, USA). Aliquots of 10 μL were first applied to a CarboPacSA10-4 μm column, and subsequently analyzed on a Dionex ICS5000+Reagent-Free HPLC System equipped with a CarboPac SA10-4 μm GuardColumn (4×50 mm). Elution of separated molecules was with an isocratic25 mM KOH run over 20 min. After baseline subtraction and employing HPLCgrade pure carbohydrates as reference standards [D-(+)-glucose,D-fructose, D-(+)-maltose maltotriose], carbohydrates were quantified bypeak area integration. The results are shown in FIG. 7 .

Example 5 Fermentation

The wort, prepared as described in Example 3, is boiled in the presenceof hops or hop extract and the fermentation process is commenced throughthe conventional inoculation of the extract with an appropriate brewingyeast strain. Fermentation, beer filtration and bottling are performedaccording to traditional protocols.

Small-Scale Brewing

The example compares beer prepared from two varieties of barley, treatedaccording to the methods described herein with beer prepared fromunmalted barley treated with a commercially available brewing enzymesmix. The wort and final beers were analysed and compared to a commercialreference lager beer (denoted “Reference” herein) when possible. Thedata on the reference lager beer were obtained separately from othersources.

Material

Unless otherwise stated the material was used as is.

Trial Trial Trial Trial 1 2 3 4 Reference Barley Hull-less 01 Hulled 02Hulled 02 Hull-less 80% malt Variety 01 20% barley Treatment at 90 L/hair at 90 L/h air — — Malt prior to 24 h WA 24 h WA prepared by milling24 h A 24 h A standard procedure

Milling

Treated material Untreated material Milled on a household Milled onMULTICRACKER juicer (Omega J822C)

Brewing

Brewing was done under standard conditions using a grits/water ratio of1 to 4.

Treated Untreated Untreated material material material (trial 1 and 2)(trial 3) (trial 4) Reference pH 5.20 5.70 5.70 Typically 5.20 EnzymeUltraflo ® Max, OndeaPro ®, OndeaPro ®, Ultraflo ® Max, addition 0.2g/kg DM 2.0 g/kg DM 2.0 g/kg DM 0.08 g/kg DM Attenuzyme ® Attenuzyme ®Flex, 1.0 g/kg Core, 0.2 g/kg DM DM

The germinated barley, the untreated material and/or the malt was mashedin using a standard mashing programme with a 2 step saccharification inthe presence of the indicated enzymes.

Ultraflo® Max, Attenuzyme® Flex, Attenuzyme® Core and OndeaPro® areavailable from Novozymes, Denmark. According to manufacturer:

-   -   Ultraflo® Max comprises β-glucanase (700 EGU/g) and xylanase        (250 FXU/g)    -   Attenuzyme® Flex comprises glucoamylase (400 AGU/g) and        pullulanase (80 PUN/g) according to product sheet from        manufacturer    -   Attenuzyme® Core comprises glucoamylase (1600 AGU/g)    -   OndeaPro® comprises β-glucanase, Xylanase, α-amylase,        Pullulanase (637 PUN/g), Protease and Lipase.

When determining the activity of Attenuzyme® Flex as described inExample 2 it was found that an amount of Attenuzyme® Flex was usedcorresponding to 16243 mU barley limit dextrinase activity per g enzymesolution. Furthermore, it was found that the combined glucoamylase andα-amylase activity was 628,863 U per g enzyme solution.

The cold wort was collected after standard lautering and and wortboiling with hops added at the start of boiling.

The original extract was adjusted with tap water to achieve a finalplato (in % P) of 11.5 after boiling and evaporation and the colour ofwort was adjusted to achieve a colour similar to the colour of thereference beer.

Fermentation

-   -   Brewers Clarex® (available from DSM) was added to the cold wort        at 0.1 g/kg DM    -   wort was pitched with 8E6 cells/ml of lager yeast (S.        pastorianus),    -   pitched wort was aerated with air for 30 min    -   fermentation was carried out in a pressure-less fermentation        tank at 15° C. until end of fermentation    -   end-fermented beer was kept at 4° C. until transfer to a tank

Transfer to Tank

-   -   beer with less than 5E5 cells/mlin suspension was transferred to        a tank    -   the tank was flushed with CO₂ to 0.5 bar before and after        filling    -   CO₂ was added to an overpressure of 0.5 bar and beer was kept at        4° C. until filtration

Filtration

-   -   beer was filtered through three layers of depth filter sheets    -   1.2 bar of CO₂ pressure were applied to beer in the tank after        filtration    -   beer was kept at 4° C. until packaging

Packaging

The beer was packaged in 33 cl bottles and kept at 4° C. for final testsand sensory evaluation. Analysis results

Sugars in Wort Before Fermentation

The concentration of total fermentable sugars was determined essentiallyas described in Example 4 and the results are shown in Table 5. Theglucose levels are noticeably higher in both brews prepared according tothe methods of the invention compared to the brew prepared from unmaltedbarley.

TABLE 5 Trial 1 Trial 2 Trial 3 Trial 4 Reference g/100 g/100 g/100g/100 g/100 g/% P g/% P g/% P g/% P g/% P wort % wort % wort % wort %wort % Sucrose 0.008 1.9 0.016 1.8  0.019 2.5 0.021 2.2 0.024 3.3Glucose 0.202 47.4  0.497 56.3   0.054 7.1 0.046 4.8 0.333 46.1 Fructose 0.012 2.8 0.014 1.5 0   0.0 0.013 0.016 2.2 Maltose 0.193 45.3 0.338 38.3   0.533 70.8  0.651 68.7  0.312 43.2  Maltotriose 0.011 2.60.017 1.9  0.147 19.5  0.217 22.9  0.037 5.1

Free Amino Nitrogen and β-Glucan

The concentration of free amino nitrogen (FAN) in the wort prior tofermentation as well as in the final beer were determined according tothe ThermoFisher, Gallary Beermaster standard protocol for FAN,Colorimetric method. Typical values of free amino nitrogen (FAN) in wortare 200 mg/L. FAN is important for good yeast viability duringfermentation. In general, FAN levels which are high enough to obtaingood yeast viability are desirable. The results re FAN in wort beforefermentation is shown in Table 6a and in beer in Table 6b.

β-glucan is usually degraded during conventional malting. Too highlevels of β-glucan is undesirable, because this may cause problematicfiltration. The level of β-glucan in wort prior to fermentation as wellas in beer were determined using the “Beta-Glucan (High MW)” kit fromThermo Scientific according to manufacturer's instructions and theresults are shown in Table 6a (wort prior to fermentation) and Table 6b(beer).

TABLE 6a Wort prior to fermentation Trial Trial Trial Trial mg/L 1 2 3 4Reference FAN 265 177 104 118 219 beta-glucan 143 107 86 151 73

TABLE 6b beer Trial Trial Trial Trial mg/L 1 2 3 4 Reference FAN 116 3650 55 104 beta-glucan 60 50 44 58 46

Amino Acids in Wort Before Fermentation

The concentration of all standard amino acids in the wort prior tofermentation were determined using Waters AccQTag Ultra kit followingthe procedure described therein. The result on amino acids in wortbefore fermentation are shown in Table 7.

In particular the concentration of valine before fermentation isimportant. The more valine is present in the wort, the lower is thelikelihood of “undesired Diacetyl” formation during fermentation—andthereby longer DA resting time. Valine concentration is 5-fold (Trial 1)and 2-fold (Trial 2) higher in wort prepared according to the inventioncompared to Trial 3.

TABLE 7 Trial Trial Trial Trial mg/L wort 1 2 3 4 Reference Histidine 2030 17 23 56 Asparagine 119 82 77 100 Serine 8 44 26 43 67 Glutamine 7945 10 16 Arginine 92 92 59 77 144 Glycine 58 34 18 21 36 Aspartic acid 954 45 53 79 Glutamic acid 87 58 36 67 72 Threonine 81 44 26 33 77Alanine 193 103 45 58 113 Proline 317 105 38 70 Cysteine 0 4 0 0 Lysine113 77 51 59 104 Tyrosine 94 56 36 45 116 Methionine 37 24 17 21 42Valine 153 71 31 50 132 Isoleucine 80 43 16 28 79 Leucine 214 109 52 69181 Phenylalanine 139 75 39 47 144 Tryptophane 47 32 25 30 Total aminoacids 1960 1212 681 907

Various brewing and fermentation key number were determined and comparedto the reference. The results are shown in Table 8. It is notable thatbeers prepared by the methods of the invention had significantly lowerdiacetyl levels. It is generally preferred that the diacetyl levels areas low as possible.

TABLE 8 Trial Trial Trial Trial 1 2 3 4 Reference Original extract, % P11.4 11.5 11.7 11.5 10.1 RDF, % 70.8 70.5 74.9 72.5 70.3 pH 4.06 3.904.21 4.15 4.25 Alcohol, vol % 5.2 5.2 5.6 5.4 4.54 Colour, EBC units 5.48.0 5.4 7.1 6.4 Bitterness 14 14 16 18 16 Diacetyl, ppb 14 15 27 28 22SO₂, mg/L 1 1 1 1 3

Sensory Panel Evaluation

All the beers prepared as described in this Example were subjected toevaluation by a sensory panel. The total flavour score for all of themwas acceptable. One difference between the different beers was that thebeer from Trial 3 had the score “noticeably” for the flavour “soapy,fatty, diacetyl, oily rancid”, whereas the beer from Trial 2 (preparedaccording to the invention) only had the score “slightly” for thisflavour.

Example 6

Steeping of Wheat:

4 commercially available wheat varieties (1-Benchmark, 2-Creator,3-Pistoria, 4-Sheriff) were sanitized for 1 h in 0.1% hypochloran, thensteeped and germinated by incubation in a tank for 24 h or 48 h in tapwater containing 1000 nM GA3 and 0.01% antifoam agent (Sigma 204). Theincubation was performed at 25° C., and 90 l/h air was lead through thewater from the bottom of the tank during the entire incubation.

After incubation the α-amylase, β-amylase and limit dextrinase activitywas determined essentially as described in Example 2. The results areshown in FIG. 9 . After 48 h incubation the α-amylase and the limitdextrinase activity is significantly increased.

Example 7

Steeping of Hulled Barley:

8 commercially available barley varieties (1—Alexis, 2—Chief, 3—Chill,4—Paustian, 5—Planet, 6—Prestige, 7—Quench, 8—Tipple) were sanitized for1 h in 0.1% hypochloran, then steeped and germinated by incubation in atank for 24 h or 48 h in tap water containing 1000 nM GA3 and 0.01%antifoam agent (Sigma 204)., The incubation was performed at 25° C., and90 l/h air was lead through the water from the bottom of the tank duringthe entire incubation.

After incubation the α-amylase activity was determined essentially asdescribed in Example 2. The results are shown in FIG. 10 . After 48 hincubation the α-amylase activity is significantly increased.

Example 8 Peeling of Hulled Barley:

Hulled barley (Hulled 02) was peeled by mechanical sandpaper treatmentfor 1, 2, 4 or 16 minutes in order to partially remove the husk. Thetreatment resulted in 1, 2, 3 or 5 weight loss. The peeled barleykernels were sanitized for 1 h in 0.1% hypochloran, and steeped andgerminated by incubation in a tank for 24 h or 48 h in tap watercontaining 1000 nM GA3 and 0.01% antifoam agent (Sigma 204). Theincubation was performed at 25° C., and 90 l/h air was lead through thewater from the bottom of the tank during the entire incubation.

After incubation the α-amylase, β-amylase and limit dextrinase activitywas determined essentially as described in Example 2. The results areshown in FIG. 11 . Peeling barley kernels to a weight loss of 2% resultsin significantly induced α-amylase and limit dextrinase activities.

Example 9

Air Rest on Hull-Less and Hulled Barley:

Hull-less (Hull-less 01) and hulled (Hulled 02) barley were sanitizedfor 1 h in 0.1% hypochloran, and steeped and germinated according todifferent steeping regimes.

WA=Water/Air:

Incubation in a tank in tap water containing 1000 nM GA3 and 0.01%antifoam agent (Sigma 204) at 25° C., while 90 l/h air is lead throughthe water from the bottom of the tank during the entire incubation.

A=Air

Incubation of moist cereal grains in a tank. During the entireincubation 90 l/h air is lead through the moist cereal grains from thebottom of the tank. Incubation performed at 25° C.

The cereal grains were incubated in WA or WA and A for the timesindicated in FIGS. 12A-12B, and the results are shown in FIGS. 12A-12B.FIG. 12A shows the result for Hull-less 01 and FIG. 12B shows the resultfor Hulled 02. 24 h incubation in water under aeration followed by 24-32h incubation without water, but still under aeration results in cerealgrains with very high enzyme activities.

Example 10

Germinated barley was prepared essentially as described above inExample 1. More specifically, barley kernels of varieties Hull-less 01and hulled 02 were sanitized with 0.1% hypochloran wash for 1 h, thensteeped and germinated by incubation for 48 h in tap water containing1000 nM GA and 0.01% antifoam agent. The incubation was performed at 25°C., and 90 l/h air was lead through the water from the bottom of thetank during the entire incubation. The germinated barley was freezedried and weighed. The formed rootlets were removed using old Munichequipment and the germinated barley was weighed again. The difference inmass before and after removal of rootlets, were considered the mass ofthe rootlets. The weight of 4 different samples was determined, but onlythe 3 first are included, because the last sample contained dust. Theresults are shown in Table 9.

TABLE 9 Hull-less 01 Hulled 02 Mass of Mass of Mass Mass rootlets MassMass rootlets before (g) after (g) (g) before (g) after (g) (g) 1 99.864 98.822  1.042 99.882  99.387  0.495 2 100.024 99.135  0.88999.871 99.47  0.401 3  98.599 97.629  0.97 99.704  99.282  0.422 average 0.97  0.44 stdev  0.08  0.05

Barley kernels from the same batch of hull-less 01 and hulled 02 werealso steeped and germinated for 96 h by standard methods. The germinatedbarley was freeze dried and weighed and the rootlets were removed usingold Munich equipment. After rootlet removal the barley was weighed againand the difference in mass before and after removal of rootlets wasconsidered the mass of the rootlets. The results are shown in Table 10.

TABLE 10 Hull-less 01 Hulled 02 Mass of Mass of Mass Mass rootlets MassMass rootlets before (g) after (g) (g) before (g) after (g) (g) 1 95.16787.026  8.141 96.343 89.867  6.476 2 95.281 87.046  8.235 95.602 89.1026.5 3 95.318 87.113  8.205 95.338 89.072  6.266 average  8.19  6.41stdev  0.05  0.13

Table 11 shows a comparison between mass (in g) of rootlets of thebarley germinated by the methods of the invention (48 h WA) and inbarley steeped by conventional methods (Malting 96 h). It is evidentthat the barley germinated by the methods of the invention havesignificantly reduced rootlet formation. FIG. 13 shows the weight lossafter rootlet removal in %.

TABLE 11 Hullled 02_48 hWA 0.44 0.05 Hull-less 01_48 hWA 0.97 0.08Hulled 02_Malting 96 h 6.41 0.13 Hull-Less 01_Malting 96 h 8.19 0.05

Example 11

Nitrosamine NDMA is formed particular in roots during kilning of malt(Wainwright (1986) J Inst Brew 92 73-80).). As described above inExample 10, an advantage of the germinated cereal grains prepared by themethods of the invention is that they comprise less roots compared toordinary green malt. NDMA content in contemporary malt is low, howeverit may be advantageous to reduce the levels even further.

NDMA content was analyzed in barley, in germinated barley grainsprepared according to the methods of the invention (denoted “Malt 1a” inthis Example) and in three industrially produced malts. The industriallyproduced malts had all been treated to remove rootlets by standardmethods.

The barley-1, Malt-1a and Malt-1b were all prepared from the same batchof a hulled barley variety (Hulled 02), while the two other malt samplesMalt-2 and Malt-3 both derive from other batches of barley. The Malt-1awas produced essentially as described above in Example 1. Thus barleykernels were sanitized with 0.1% hypochloran wash for 1 h, then steepedand germinated by incubation for 48 h in tap water containing 1000 nM GAand 0.01% antifoam agent. The incubation was performed at 25° C., and 90l/h air was lead through the water from the bottom of the tank duringthe entire incubation.

The germinated barley was freeze dried before analyzing NDMA content byGC-MS. The results are shown in FIG. 14 . The analysis clearly indicatethat less NDMA is present in Malt-1a compared to malts produced bystandard malting including kilning, even though the standard had beensubjected to deculming.

Summary

The challenges of future water and energy shortages must be addressed ina socially, economically and environmentally responsible manner. In thatrespect, the present invention contributes to the long-termsustainability of beer production in terms of reduced water and energyusage. Through elimination of the kiln drying process, combined with thedirect integration of the steeping and germination into the brewingprocess, application of the methods of the instant invention greatlyreduce the input and running costs of beer manufacture.

The instant invention may contribute to lowering input costs andreducing the environmental pressure on the malting and brewingindustries in numerous ways, including:

-   -   The steeping and germination processes that currently take        several days to complete may be completed much faster    -   The steeping and germination processes may be carried out in a        single vessel at a single location    -   The traditional air rest and second steep steps of the malting        process may be eliminated    -   The processes may reduce water consumption, for example by up to        40%    -   Expensive heating costs to kiln-dry the malt may be eliminated    -   Expensive transport costs to shift malt from the malthouse to        the brewery may be eliminated    -   Equipment and plant required to perform the methods of the        invention may be compatible with existing equipment in brew        houses and will therefore not require large new capital        expenditure.

LITERATURE CITED

-   Bamforth C (2009) In: Beer: Tap into the art and science of brewing.    Oxford University Press.-   Briggs D E (1998) In: Malts and Malting. Blackie &, Professionals.-   Burton R A, Wilson S M, Hrmova M, Harvey A J, Shirley N J, Medhurst    A, Stone B A, Newbigin E J, Bacic A and Fincher G B (2006) Cellulose    Synthase-like CsIF Genes Mediate the Synthesis of Cell Wall    (1,3;1,4)-β-D-Glucans. Science 311, 1940-1942.-   Burton R A, Jobling S A, Harvey A J, Shirley N J, Mather D E, Bacic    A, Fincher G B (2008) The genetics and transcriptional profiles of    the cellulose synthase-like HvCsIF gene family in barley (Hordeum    vulgare L.). Plant Physiol., 146: 1821-1833.-   Burton R A, Collins H M, Kibble N A J, Smith J A, Shirley N J,    Jobling S A, Henderson M,

Singh R R, Pettolino F, Wilson S M, Bird A R, Topping D L, Bacic A andFincher G B (2011) Over-expression of specific HvCsIF cellulosesynthase-like genes in transgenic barley increases the levels of cellwall (1,3;1,4)-β-D-glucans and alters their fine structure. PlantBiotechnol. J., 9: 117-135.

-   Fincher G B (2011) Biochemistry, Physiology and Genetics of    Endosperm Mobilization in Germinated Barley Grain. In: Barley:    Production, Improvements and Uses. Ed. Ullrich S E, Wiley-Blackwell,    Chapter 14, pp 449-477.-   Smith A M, Zeeman S. C, Smith S M (2005) Starch Degradation. Annual    Review of Plant Biology 56: 73-98.-   Taketa, S., Yuo, T., Tonooka, T., Tsumuraya, Y., Inagaki, Y.,    Haruyama, N., Larroque, O., and Jobling, S. A. (2011) Functional    characterization of barley beta-glucan-less mutants demonstrates a    unique role for CsIF6 in (1,3;1,4)-β-D-glucan biosynthesis, J. Exp.    Bot. 63, 381-392.

1. A method for producing an aqueous extract of a cereal, said methodcomprising the steps of: A. providing germinated grains of a cerealhaving a water content of at least 20% with the proviso that said cerealgrains have not had a water content below 20% at any time subsequent togermination, wherein the cereal is a barley characterized by carrying amutation in the gene encoding MMT, and wherein the entire step ofgerminating said germinated grains did not exceed 72 hours (h); B.finely dividing said germinated grains, while said germinated grainshave a water content of at least 20%; and C. preparing an aqueousextract of said finely divided germinated grains, thereby producing anaqueous extract of the cereal.
 2. The method according to claim 1,wherein the germinated grains have an α-amylase activity of at least 4U/g cereal grain on a dry weight basis.
 3. The method according to claim1, wherein the germinated grains contain at the most 4 gram (g) rootlets(dry matter) per 100 g germinated cereal grains (dry matter).
 4. Themethod according to claim 1, wherein the cereal is a barleycharacterized by carrying a mutation in the gene encoding LOX-1 andcarrying a mutation in the gene encoding MMT.
 5. The method according toclaim 1, wherein the cereal is a barley characterized by one or more ofthe following: A. carrying a mutation in the gene encoding a β-glucansynthase B. carrying a mutation in the gene encoding LOX-1 C. carrying amutation in the gene encoding LOX-2; and D. carrying a mutation in thegene encoding DELLA
 6. The method according to claim 1, wherein thegerminated grains have been prepared by a step of germination comprisingincubating said grains in an aqueous solution while O₂ is passed throughsaid aqueous solution.
 7. The method according to claim 1, wherein themethod comprises the steps of: a. providing grains of a cereal; b.subjecting the cereal grains to a step of germination thereby obtaininggerminated grains, wherein said step of germination comprises incubatingsaid grains in an aqueous solution until the grains have a water contentof at least 30%, wherein at least 2 L O₂ per kg dry weight cereal grainsis passed through said aqueous solution per h; and at least one step ofair-rest, wherein said cereal grains are incubated in air; c. finelydividing said germinated grains, while said germinated grains have awater content of at least 20%, with the proviso that said cereal grainsdo not have a water content below 20% at any time between steps b) andc); and d. preparing an aqueous extract of said finely dividedgerminated grains.
 8. The method according to claim 7, wherein the stepof germination comprises the steps of a) incubating said grains in saidaqueous solution, wherein at least 2 L O₂ per kg dry weight cerealgrains is passed through said aqueous solution per h; b) air-rest,wherein said cereal grains are incubated in air; c) incubating saidgrains in said aqueous solution, wherein at least 2 L O₂ per kg dryweight cereal grains is passed through said aqueous solution per h.; andd) air-rest, wherein said cereal grains are incubated in air.
 9. Themethod according to claim 7, wherein the air-rest is in the absence ofaqueous solution.
 10. The method according to claim 6, wherein the stepof incubating said grains in an aqueous solution under aeration isperformed in the range of 16 to 30 h.
 11. The method according to claim8, wherein the step of incubating said grains in an aqueous solutionunder aeration is performed in the range of 20 to 30 h or in the rangeof 22 to 26 h.
 12. The method according to claim 8, wherein the step ofincubating said cereal grains in air is performed in the range of 18 to50 h.
 13. The method according to claim 8, wherein the step ofincubating said cereal grains in air is performed in the range of 18 to38 h or in the range of 22 to 35 h.
 14. The method according to claim 1,wherein the entire step of germination does not exceed 54 h.
 15. Themethod according to claim 1, wherein the method is performed in one ormore apparatuses suitable for performing the method.
 16. The methodaccording to claim 6, wherein said O₂ is contained in a gas mixture,wherein the gas mixture is atmospheric air.
 17. A method for producing abeverage, said method comprising the steps of: i. preparing an aqueousextract by the method according to claim 1; and ii. processing saidextract into a beverage.
 18. The method according to claim 17, whereinstep ii comprises the steps of: a. heating said aqueous extractoptionally in the presence of hops or hops extract; b. cooling theaqueous extract; and c. fermenting said aqueous extract with yeast,thereby producing a fermented beverage.
 19. The method according toclaim 17, wherein the beverage is lager beer.